Application of Bioelectrochemical Technique in Energy Recovery and CH4 Control From Constructed Wetland (CW) and Associated Biochemical Mechanisms

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The main focus is to further explore the effect of plant roots location at the electrode, plant species on CH 4 emissions, bioelectricity generation and the mechanism underlying competition between electrogenesis and methanogenesis at the anode. The results showed that the operation of MFC effectively reduced the CH 4 emissions and promoted COD removal rates. CH 4 emission was significantly higher in open circuit (6.2 mg m -2 h -1 ) than in closed circuit reactors (3.1 mg m -2 h -1 ). Plant roots at the cathode had the highest electricity generation and the lowest CH 4 emissions. The highest power generation (0.49 V, 0.33 w m -3 ) and the lowest CH 4 emissions (2.3 mg m -2 h -1 ) were observed in the reactors where Typha Orientalis was planted with plant roots at the cathode. The role of plants in strengthening electron acceptor was greater than that of plant rhizodeposits in strengthening electron donors. q-PCR and correlation analysis indicated that the mcr A genes and CH 4 emissions were positively correlated ( r=0.98, p<0.01 ), while no significant relationship between CH 4 emissions and pmoA genes was observed. More nanowires, which are conductive to electron transfer, were found when plant roots were in cathode by scanning electron microscope (SEM). Illumina sequencing revealed that more abundant exoelectrogens and denitrifying bacteria ( Geobacter , Desulfobulbu, Nitrospira and Anaerolinea) were observed when plant roots located in cathodes. Strictly acetotrophic archae (Methanosaetaceae) were likely main electron donor competitors with exoelectrogens. In addition, plant species played a more important role in CH 4 emissions and electricity generation than the plant roots location at the electrode. Therefore, it is necessary to strengthen plant configuration to reduce CH 4 emissions, so as to promote sustainable development of wastewater treatment. Biotechnology and Bioengineering CW-MFC CH4 control Bioelectricity generation Plant root location Biochemical mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction In the context of global warming and the increasingly significant impact of human activities on the environment, greenhouse gas emission reduction, low-carbon economy and low-carbon cities are the research hotspots nowadays [ 20 ] , [ 34 ] . Global climate change caused by the increase of greenhouse gas emissions in the atmosphere has become the focus of human concern, and is also a huge challenge facing the sustainable development of the world economy. Methane is an important greenhouse gas, which is considered to be one of the most important greenhouse gases after CO 2 . Its greenhouse effect is 20–30 times that of CO 2 [ 35 ] , which contributes about 20% of the global climate directly drive [ 27 ] . Methane has become an important part of global "carbon reduction". Constructed wetland (CW) is considered as a reliable wastewater treatment technology, which has the advantages of less investment, less operation cost, easy maintenance and stable effluent quality [ 29 ] , which plays an important role in non-point source pollution control. However, constructed wetlands will release greenhouse gases such as CO 2 , CH 4 and N 2 O during operation, which will have a negative impact on the environment. Studies showed that constructed wetlands emit 2 to 10 times more greenhouse gases than natural wetlands [ 15 ] , its role in global warming cannot be ignored. In recent years, scholars have also carried out research on methane emission reduction in CWs. In general, methane emission control in CWs can be divided into two categories: first, macro control, including physical control methods such as aeration [ 17 ] , [ 24 ] , Chemical methods (e.g. REDOX potential control, iron ion control, chemical substance control) [ 13 ] , Hydrologic method (influent pattern and water level variation) etc [ 7 ] . Another method is micro-control, such as molecular biological methods [ 9 ] and nano-control [ 5 ] . So far, however, study on the microscopic control of methane emissions is limited. In particular, the literature on methane control by microbial or bioelectrochemical method is quite limited. Microbial fuel cell (MFC) is a device that using electrogenic microorganisms to directly convert chemical energy of organic matter into electrical energy. In the past 10 years, a lot of work has been done to improve the electrical performance of MFC, wastewater treatment efficiency, reactor configuration and electrode materials [ 12 ] , [ 37 ] , [ 41 ] . But it is a pity that its electricity generation efficiency is still very low and its engineering application is limited. Therefore, the development and application of MFC in the academic community caused controversy and doubt [ 26 ] . In fact, we can change our thinking and use MFC as a tool rather than as a research purpose. Due to the similar operating conditions of microbial fuel cell and constructed wetland, the integration of MFC with CW technology becomes possible. Since CW-MFC integrated technology was first proposed in 2012 [ 39 ] , it has been proved in recent years that CW-MFC Technology can greatly improve the sewage treatment efficiency and electricity generation performance [ 22 ] , [ 38 ] . Theoretically, there exists a competitive relationship between electrogenic microorganisms and methanogens in the anodes of MFC and CW, which can inhibit methane emission. Our previous study confirmed that methane emission could be inhibited by running MFC [ 43 ] . However, the research on the regulation of methane emission in CW by bioelectrochemical method (MFC) is still inadequate and very limited. In addition, there are still many scientific problems to be further explored. It is not clear, for example, whether the type of plant and the location of plant root system on the electrode has an effect on methane emissions, how much, and how it works. In the current CW-MFCs studies, the plant roots were mostly placed at the anode [ 21 ] , [ 33 ] , while in some studies, the plant roots were mostly placed at the cathode. Theoretically, placing them at the anode would provide organic matter for electricity generation, while placing them at the cathode would increase the number of electron receptors needed to generate electricity. However, it has not been reported whether the plant roots placed at the anode or the cathode are more conducive to electricity generation and methane emission. Therefore, this study aims to further explore the influence mechanism of MFC on CW methane emissions, wastewater treatment and electricity generation based on the above problems. The influence of the electrode position of plant root system on methane emission and electrical performance and associated biochemical processes were mainly explored. The mechanism underlying competition between electrogenesis and methanogenesis at the anode was investigated. The systematic elucidate of the biological process involved in the integrated system (CW-MFC) will provide a theoretical basis for CH 4 control in constructed wetlands and promote the sustainable development of wastewater treatment. 2. Materials And Methods 2.1. MFC-CW construction and operation Eight polypropylene plastic circular columns with the height of 0.55 m and the diameter of 0.18 cm were adopted as the main reactors to simulate the vertical flow CW-MFC system. Two plant species ( Typha orientalis, Cyperus alternifolius ) were planted in anodes and cathodes, respectively. In CW-MFCs planted with Typha Orientalis , plant roots located at the cathode and anode were named as P1-Ca and P1-An, respectively. In CW-MFCs planted with Cyperus alternifolius ), plant roots located at the cathode and anode were named as P2-Ca and P2-An, respectively. The other four reactors operated under open circuit as a control, marked as CK1, CK2, CK3 and CK4. From the bottom to the top of the reactor are: the first layer is 10 cm thick gravel, the second layer is 10 cm thick granular activated carbon GAC (anode layer), the third layer is 20 cm thick gravel, and the fourth layer is 5 cm thick granular activated carbon (cathode layer). Stainless steel wire mesh (12 cm in diameter) is placed in the middle of the anode layer and the cathode layer respectively to collect and transfer electrons. The cathode and anode are connected by titanium wire, and the external copper wire is connected with the resistance box to form a closed loop. The schematic diagram of the CW-MFCs is shown in Fig.1, the control reactors in open circuit are not shown. In this study, the selection of electrode materials, the setting of electrode distance and the configuration were according to the reported literature [4] , which has good power generation, wastewater treatment capacity and economic feasibility. Activated sludge was taken from a local wastewater treatment plant. The aerobic activated sludge was inoculated at the cathode of CW-MFCs and the anaerobic activated sludge was inoculated at the anode of CW-MFCs. The reactor operated in a continuous flow mode with water inflow at the bottom. The synthetic wastewater was composed as follows: KCl 130.0 mg/L, Na 2 HPO 4 ·12H 2 O 275.0 mg/L, NH 4 Cl 133.7 mg/L, NaHCO 3 313.0, MgSO 4 ·7H 2 O 25.0 mg/L, CaCl 2 15.0 mg/L, NaH 2 PO 4 ·2H 2 O 497.0 mg/L, trace elements 0.1 ml/L. The influent COD was controlled at 300 mg/L. The hydraulic residence time was set according to the experimental requirement. The reactors were operated at 26 ± 2 °C. 2.2 COD measurement and CH 4 fluxes The chemical oxygen demand (COD) was determined by potassium dichromate method. The methane was collected by a static chamber method and analyzed by a gas chromatograph (PerkinElmer GC Clarus 680). The injection temperature was 85°C, the split ratio was 20:1, the column temperature was 75 °C, and the gasification temperature was 250 °C. The content of CH 4 was calculated by the linear relationship between the peak area and the known content. The methane emission flux was calculated by the following formula: In the equation: E methane -- methane emission flux, g/m 2 /h; ρ--The density of methane in standard condition, 714.3 g/m 3 ; V -- Closed volume of collected gas, m 3 ; A -- Area of electrode surface, m 2 ; dc methane/dt -- Change in methane gas in a given time, mg/ mL /h; T -- average temperature 2.3 Bioelectricity generation monitoring Bioelectricity generation performance was measured under the resistance of 1000 Ω by data acquisition system (model 2700, Keithley Inc.). The voltage data was recorded every 5 minutes. The current value is obtained according to voltage and resistance (I=U/R). Subsequently, power density (P, W/m 3 ) and current density (J, A/m 3 ) were calculated according to the anode volume. The current density and power density were calculated as J = U/RA and P = IU/A, respectively. Where, I is the current, U is the voltage, R is the resistance and A is the working volume of the anode. The polarization curve was determined by changing the external electrical resistance from 50 to 90,000 Ω at the end of experiment. 2.4 DNA extraction and Illumina sequencing The total DNA of samples was extracted by a Power Soil DNA Isolation Kit 178 (MOBIO Laboratories Inc., USA) according to the manufacturer’s instructions. Bacterial 16S rDNA and archael gene for pyrosequencing were amplified by PCR. The primers BAC27F (5'-AGAGTTTGATCCTGGCTCAG-3'), BAC1492R (5'-GGTTACCTTGTTACGACTT-3' and the primers ARC25F (5'-CYGGTTGATCCTGCCRG-3'), ARC958R (5'-YCCGGCGTTGAMTCCAATT-3') were used for bacterial and archaea, respectively. The products of PCR were loaded on 1.5% agarose gel and the gel was purified with Gel Extraction Kit (OMEGA Bio-tekInc, USA). The DNA concentration and quality were determined by real-time PCR (FTC-3000™), subsequently the amplicons were further used for Illumina MiSeq sequencing. All the raw reads were analyzed by combining different programs from the quantitative insights into microbial ecology (QIIME) after sequencing. When the low quality adaptors, primers, barcodesand reads were eliminated, the remaining 16S rRNA sequences were clustered into OTUs using UCLUST with the distance of 0.03. 2.5 Quantitative PCR ( q-PCR) of functional genes The function genes for methanogens and methane-oxidizing bacteria were determined by q-PCR on an ABI 7500 real-time PCR system instrument (Applied Biosystems, USA). Primers mcrA-f (5′-GGTGGTGTMGGATTCACACARTAYGCWACAGC-3′) and mcr A-r (5′-TTCATT GCRTAGTTWGGRTAGTT-3′) were used for mcr A gene amplification. Primers A189-f (5′-GGNGACTGG GACTTCTGG-3′) and mb661-r (5′-CCGGMGCAACGTCYTTACC-3′) were used for pmo A gene amplification. The reaction volume, the reaction temperature, the reaction procedure of PCR amplification and the plasmid curve were plotted according to our previously reported methods [42] . 2.6 Scanning electron microscope (SEM) analysis of bioanode The morphology of anode microorganism was imaged using SEM (JSM-5610, Japan). The samples were treated as follows: A small amount of sample was placed in a 5 mL centrifuge tube, 2.5% glutaraldehyde was added, and fixed in the refrigerator for 1.5 hours. And then rinsing with phosphate buffer solution of 0.1mol/L (pH=6.8) for three times. The samples were dehydrated with 50%, 70%, 80% and 90% ethanol in turn. Rinse with a mixture of anhydrous ethanol and isoamyl acetate at volume ratio of 1:1 for 15 minutes, then rinse with pure isoamyl acetate for 15 minutes. The dried sample was pasted on the sample table of SEM and coated with Au film of 1500 nm thickness on the sample surface. Finally, the samples were observed under SEM. 2.7 Statistical analysis Statistical analyses were conducted using SPSS software (version 21.0) (SPSS Inc., Chicago, USA). Significance analysis was used to identify differences between experimental groups or control groups by one-way ANOVA at p <0.05.The R software package and Origin (version 8.0) were used for bioinformatic analysis graphical works. 3. Results And Discussion 3.1 CH 4 emissions from CW-MFCs After four weeks of batch operation, the voltage in all reactors reached a steady state. The reactors then operated in a continuous flow mode and began recording data. During the experiment, the plant death in two reactors led to abnormal data (CK3, CK4), so the data of these two reactors would not be recorded in the subsequent experiment. So there are six reactors running stably. As shown in Fig. 2 a, CH 4 emissions from all reactors showed a downward trend with the increase of hydraulic residence time (HRT). CH 4 emission was significantly higher in open circuit than in closed circuit. When HRT = 1, the average CH 4 emissions of CK1 and CK2 reached 6.1 mg m − 2 h − 1 and 6.4 mg m − 2 h − 1 (Fig. 2 b), indicating that MFC operation in constructed wetlands can effectively reduce methane emission. When plant roots were located at the anode, methane emission was significantly higher than that at the cathode (P1-an = 3.9 mg m − 2 h − 1 , P2-an = 4.5 mg m − 2 h − 1 ). CH 4 emissions was lowest when plant roots were located at the cathode, with an average of 2.3 mg m − 2 h − 1 found in P1-Ca. Statistical analysis showed that both plant and root location had an impact on methane emission, but the impact of plant root location on methane emission was more significant ( P < 0.01). CH 4 emissions from the reactor planted with Typha Orientalis were lower than those of the reactor planted with Cyperus Alternifolius both at the cathode and anode. With the increase of HRT, methane emission in CW-MFC tends to decrease, which is consistent with previous research conclusion of CH 4 emissions in CW [ 16 ] . Although a lot of work has been done on the role of plants in CWs, consensus has been reached in the academic circle that plants have little impact on the wastewater treatment efficiency of constructed wetlands. The contribution of plants to sewage treatment is about 10%, while the fillers, matrix, operation modes and other factors contribute 90% of sewage treatment efficiency [ 3 ] , [ 28 ] . However, different plant species on the bioelectricity generation and CH 4 emissions in CW-MFCs have not been fully explored. From our results, it can be seen that plants played an important role in CH 4 in CW-MFCS. Therefore, from the perspective of sustainable development of wastewater treatment, more attention should be paid to plant species selection during CW development and management. 3.2 COD removal efficiency The removal efficiency of COD was measured under different HRT. The results show that the removal efficiency of COD increases with the increase of HRT (Fig. 3 ). Except for P1-An, the COD removal efficiency was positively correlated with HRT. The removal rates of COD in close-circuit reactors were significantly higher than that of open-circuit reactors. This result indicated that the operation of MFC effectively promoted the degradation of organic matter. The average COD removal rate was about 85% in the control groups. In contrast, the average COD removal rate is more than 90% in the CW-MFC reactors. Previous studies have also shown that MFC integrated with CW could significantly promote the efficiency of sewage treatment [ 31 ] , [ 38 ] . The probable reason may be that electrochemical processes can promote the efficiency of electron transfer, thus effectively improving the degradation of pollutants [ 28 ] . When plant roots were located at the cathode, COD removal efficiency was higher than that of plant roots at the anode. In the P1-Ca reactor, the average COD removal rate reached 95.1%. Unlike the removal of COD, there was no significant difference between two plant species ( p > 0.05).This results were consistent with previous studies on the effectiveness of plants in removing pollutants from CWs [ 11 ] , [ 23 ] . 3.3 Bioelectricity performance The bioelectricity generation performances in four CW-MFCs were monitored. As shown in Fig. 4 , the average voltage generated by the four reactors varied significantly. The voltage of plant roots at the cathode was greater than that at the anode (P1-Ca = 490 mV, P1-An = 437 mV). P2-Ca = 354 mV, P2-An = 321 mV). Different from the influence of plants on COD removal efficiency, plant species had a significant influence on voltage. The voltage generated by the reactor growing Typha Orientalis was higher than that generated by the reactor planted Cyperus Alternifolius , whether the plant roots were at the anode or the cathode. This may be related to the type and amount of exudates and organic matter in the root system of different plant species [ 14 ] . The organics from plant rhizodeposits can provide more electron donors for the electrochemical active bacteria (EAB). Plant roots at the cathode have a better electrical performance than plants at the anode, probably because they have limited electron receptors in the system, and the oxygen produced by plants increases the electron receptors [ 25 ] . Thus, the electrical performance of the reactor is improved. However, the effect of the improved electron acceptor brought by the plant root at the cathode on the electric efficiency was less than that of the plant species. The polarization curve and the power density curve of four CW-MFCs were obtained by changing the external resistance from 50 Ω to 90 kΩ (Fig. 5 ). The P1-Ca produced maximum power density of 0.22 W m − 3 . The corresponding current density was 0.4 A m − 3 . The lowest power density was observed in P2-An with 0.058 W m − 3 . In the reactor planted with Cyperus Alternifolius , the power density was less than 0.14 W m − 3 . On the contrary, in the reactor where Typha Orientalis was planted, the power density was above 0.22 W m − 3 . These results indicated that plant species had a significant effect on power density in CW-MFC systems. Due to the enhanced performance of wastewater treatment and bioelectricity generation, bioelectrochemical systems (MFC and MEC) integrated constructed wetlands have gained a considerable amount of attention [ 10 ] . The enhancement of wastewater treatment and bioelectricity generation mainly emanated from the electron transfer or flow, particularly in anaerobic areas [ 25 ] . However, there are few studies on the influence of plant roots location on the electrical performance in CW-MFCs system, and the references available are still very limited. Previous study showed that plant roots located at the anode could improve the electrical performance due to root exudates and other reasons [ 36 ] . In our study, it is found that when plant roots were located at the cathode, the role of plant in increasing electron donor is greater. The existence of electron donors is conducive to improving the electrical performance [ 28 ] . However, at present, both CW and CW-MFC have low power generation, which is difficult to be applied in engineering. Inadequate electron transfer may be one of the important reasons for the low efficiency of electricity generation. Therefore, further understanding the electron transfer between electrode-anode and cathode in CW-MFCs is essential to improve the development of this integrated technology. 3.4 q-PCR of mcrA genes and pmoA genes To quantify the methanogens and methanotrophs in reactors, samples were collected from the anode and cathode for the determination of these two bacteria, respectively. The quantification of mcrA genes and pmoA genes, which encode methanogenesis and methanoxase, were investigated by q-PCR. The results showed that the number of mcrA genes in open-circuit systems was greater than that in closed-circuit systems (Fig. 6 ). The number of mcrA genes at the anode was greater than that of cathode. Meanwhile, the differences between plant species were observed. In the reactor planted with Cyperus Alternifolius , the average number of mcrA gene is 3.6× 10 4 copies mL − 1 . In contrast, in the reactor planted with Typha Orientalis , the average number of mcrA genes is 4.7× 10 4 copies mL − 1 . Correlation analysis showed that there was a significant positive correlation between the number of methanogens and CH 4 production in the reactor (r = 0.98, p < 0.01) . Unlike mcrA genes, pmoA genes increased slightly in closed-circuit systems. At the same time, the number of pmoA genes in the plant root at the cathode was greater than that in the anode. The influence of plant species on the number of pmoA genes is not obvious. Correlation analysis showed there was no significant correlation between CH 4 emissions and the number of pmoA genes, which suggests that plants not only affect CH 4 emissions by influencing methanotrophs, but also through their physiological functions, such as plant conduction. Methane emission is the net result of methane production, oxidation and transport [ 18 ] . The production and oxidation of methane are closely related to microorganisms, especially methanogens and methane-oxidizing bacteria (methanotrophs) are important biological factors that determine methane emission. However, from the results of this study, there was no significant correlation between CH 4 emissions and methanotrophs, indicating that other factors may play a key role. Plants play an important role in methane production and transport. On the one hand, the organic matter and plant rhizodeposits provided by different plants can lead to different CH 4 emissions. On the other hand, plants affect the redox potential in the rhizosphere zone and the ratesof oxygen transport via plant aerenchyma from the rhizosphere [ 6 ] , [ 42 ] . In addition, some plants have been reported to release methane under aerobic conditions [ 1 ] . Therefore, a lot of systematic studies about the influence of plant species on CH 4 emissions in the CW-MFCs are still needed, rather than the current studies on phenomena. 3.5 Bacterial and archael community analysis The bacterial community composition at phylum level is shown in Fig. 7 a. Bacteroidetes and Planctomycetes were the predominant community in CKs, which accounted for 21% − 29% of total composition. In contrast, their abundances in the other MFC-CWs were low. Chloroflexi and Proteobacteria were dominant community in close circuit MFC-CWs, which was significantly higher than that of CKs. Proteobacteria as the most abundant population in P1-CA reactor accounted for 29%, followed by Chloroflexi (15%). In addition, the abundance of Firmicutes in close circuit MFC-CW was higher than that of CKs. The community abundance of other phyla did not differ significantly in all reactors. At the genus level, the community composition of all reactors also showed significant differences (Fig. 7 b). The genera of exoelectrogens ( Geobacter, Geothrix and Desulfobulbus ) were not detected in CKs. The main community composition of CKs was Saprospiraceae and Arthrobacter . P1-Ca and P2-Ca had high abundance of exoelectrogens. The highest value (21%) of Geobacter was observed in P1-Ca. All the reactors had the nitrification and denitrification community ( Nitrospira , Brachymonas and Nitrosomonas ). In addition, Anaerolinea was found had high percentage in close circuit MFC-CWs. A high proportion of Proteobacteria was found in the reactor, which may be related to the formation of exoelectrogens and the increase of nitrification and denitrification bacteria [ 40 ] . These bacteria belong to the pylum of Proteobacteria. High abundance of Firmicutes in CC MFC-CW resulted in high electricity generation and COD removal. Firmicutes belongs to cellulolytic bacteria, which can hydrolyze cellulose derived from dead plant roots into smaller compounds like fatty acids [ 32 ] . Theses acids can then be utilized by EAB to electricity generation. Chloroflexi belongs to fermentative bacteria, can ferment small molecular saccharides to short-chain fatty acids and H 2 [ 19 ] , which can be used as electron donors for EAB. Therefore, their high proportion in CC MFC-CWs promotes the increase of current in the reactor. The common dephosphorization bacteria (Accumulibacter phosphatis and Tetrasphaera) in wastewater treatment plants were not detected, which indicated that the removal of phosphorus in the MFC-CWs was mainly through the absorption of plant rather than biological removal. The denitrification bacteria (Nitrospira, Brachymonas and Nitrosomonas), which were widely detected in the municipal sewage treatment plant, were detected in the Closed Circuit MFC-CWs, indicating that they have potential for denitrification. The Geobacter was widely reported as exoelectrogen in MFC. While the Desulfobulbus and Geothrixas were rarely reported in MFC-CW. Its role remains to be further studied. The archeal community composition is shown in Fig. 8 . Methanosaeta and Methanosarcina were the dominant archae in both cathode and anode in CK. Methanosaeta were also predominant in all the other reactors, but their relative abundance dropped around 40% in close circuit CW-MFCs. Hydrogenotrophic methanogens (Methanobacteriales and Methanococcales) showed an increased trend in all the electrical generation reactors. Methanopyrales, Thermococcales and Desulfurococcales were not detected in CKs, but they were in high percentage in electrical generation reactors. The higher values of Methanosarcina were observed in P1-Ca and P2-Ca, with 20% and 15% abundance, respectively. At the same time, about 8% of the archaea were not identified,suggesting a large number of unknown archaea. At present, there is very limited information about archaea communities and their roles in CW-MFCs. The decrease of strictly acetotrophic methanogens (Methanosaetaceae) in close circuit reactors was related the hydrogen ions formation and competition between acetotrophic archaea and bacteria [ 8 ] . On contrary, the hydrogenotrophic methanogens (Methanobacteriales and Methanococcales) tended to increase, the probable reason was that the formation of hydrogen ions and electrons during electricity generation improved the activity of hydrogenotrophic methanogens [ 30 ] . Thermococcales and Desulfurococcales are the hyperthermophilic archaea, which were seldom reported in previous studies regarding the archeal community of MFCs. Thermococcales and Desulfurococcales were reported only in plant fuel cells [ 14 ] , their role is unclear. Therefore, the role and mechanism of these hyperthermophilic archaea need to be further studied. Methanopyrales has not been reported in archaeal communities of MFCs. Desulfurococcales were reported to be autotrophic and heterotrophic. Some of them are autotrophic archae using hydrogen as the electron donor and sulfur, nitrate, nitrite and thiosulfate as the electron acceptor, some of them can also grow through organic fermentation [ 14 ] . They were predominant in P1-Ca and P2-Ca, resulting in high electricity generation. They may participate in fermentation process to produce simple material for electricity generation and methane inhibition. 3.6 SEM analysis of electrode microorganisms The morphologies of the electrode biofilm collected from reactors were scanned using SEM. As shown in Fig. 9 , various forms of microorganisms, including cocci and bacilli, were adsorbed on the electrode surface. In particular, in P1-Ca reactors, numerous types of microbes were found to be tightly attached to the electrodes including spiral, rod and spherical. Microbes on the anode are more diverse than those on the cathode. At present, the morphology of anode microorganisms has been studied intensively, but the morphology and function of cathode microorganisms have been studied less. Nanowires, circled in red, were also found on the microbial surface except P2-An. These wires originated from the cell surface and extend in different directions. Interspecific electron transport, including direct electron transport (DIET) and indirect electron transport, greatly accelerates biochemical reactions [ 44 ] . There are three types of interspecific direct electron transport: REDOX proteins, nanowires, and electron shutters (riboflavin, anthraquinone). One of the most common is nanowires. The number and mode of nanowires have an important impact on the efficiency of electricity generation [ 2 ] . In this study, the largest number of nanowires was found in the P1-Ca reactor, which may also be related to the high efficiency of electricity generation. How to enhance the interspecific electron transfer and overcome the electrolyte resistance and charge transfer resistance caused by too slow reaction rate on the electrode are the keys to improve the efficiency of CW-MFC. Therefore, more relevant studies are needed in the future. In this study, we mainly focused on the effects of plants and their roots location on CH4 emission, electricity generation and COD removal, and did not focus on the effects on electrode materials and electrode distance. In fact, electrode materials and electrode distance may have a great influence on the performance of CW-MFCs [ 25 ] , [ 28 ] . At present, there are few studies on the relationship between the configuration and performance of CW-MFC reactor. The effect of reactor configuration on CH 4 emissions has not been studied. In addition, the current research on CW-MFC is in the experimental scale, and the actual scale of the system is still in the development stage. 4. Conclusion The results showed that the operation of MFC could not only improve the wastewater treatment efficiency of CW, but also effectively reduce the methane emission, mainly through the competition between electrochemical active bacteria (EAB) and methanogenes at the anode. EAB were more advantageous than methanogens in utility of carbon sources and became the dominant bacteria group of anode. Plant roots located at the cathode are more conducive to electricity generation and CH 4 control. The enhancement effect of plant roots as electron donors in CW-MFC system was greater than that of root exudates as electron donors. At the same time, plants species played a more important role in CH 4 emissions in the CW-MFC system than the location of plant roots at the electrode. Although our study showed that running MFC in CW and plant roots located at the cathode were more conducive to energy recovery and CH 4 control, its durability of CH 4 emissions mitigation over a long period of time is still a challenge. Declarations Fundings This work was supported by the National Natural Science Foundation of China (No. 51808363) and Foundation of Chengdu Science and Technology Bureau (No. 2019-YF05-00839-SN) Authors Contributions Ke Zhang: Writing - original draft, Writing - review & editing. Xiangling Wu: Methodology, Writing - review & editing. Wei Wang: Methodology, Writing - review & editing. Hongbing Luo: Writing - review & editing. Wei Chen: Formal analysis, Writing - review & editing. Wei Chen: Data curation, Formal analysis, Writing - review & editing. Jia Chen: Writing - review & editing. Data availability Not applicable Ethics Approval This article does not contain any studies with human participants or animals performed by any of the authors. Consent to Participate Not applicable Consent to Publish Not applicable Conflict of Interest The authors declare that they have no conflict of interest. References Bruhn, D., Møller, I.M., Mikkelsen, T.N., Ambus, P., 2012. Terrestrial plant methane production and emission. Physiol Plantarum. 12, 117-124. Carlos, R., Amanda, P., Carlos, A., Pedro, C., Abraham, E.E., 2018. Microbial electrochemical technologies for wastewater treatment: principles and evolution from microbial fuel cells to bioelectrochemical-based constructed Wetlands. Water Res. 10(9), 1128-1135. Fan, L., Dippold, M.A., Ge, T., Wu, J., Thiel, V., Kuzyakov, Y., Dorodnikov, M., 2020. Anaerobic oxidation of methane in paddy soil: Role of electron acceptors and fertilization in mitigating CH4 fluxes. Soil Biology and Biochemistry, 141, 107685. He, L., Du, P., Chen, Y., Lu, H., Cheng, X., Chang, B., Wang, Z., 2017. Advances in microbial fuel cells for wastewater treatment. Renewable and Sustainable Energy Reviews, 71, 388-403. Ho, T.A., Criscenti, L.J., Wang, Y., 2016. Nanostructural control of methane release in kerogen and its implications to wellbore production decline. Sci Rep, 6, 28053. Huang, T., Liu, W., Zhang, Y., Zhou, Q., Wu, Z., 2020. A stable simultaneous anammox, denitrifying anaerobic methane oxidation and denitrification process in integrated vertical constructed wetlands for slightly polluted wastewater. Environ. Pollut. 265, 119-128. Kasak, K., Mander, Ü., Truu, J., Truu, M., Järveoja, J., Maddison, M., Teemusk, A., 2015. Alternative filter material removes phosphorus and mitigates greenhouse gas emission in horizontal subsurface flow filters for wastewater treatment. Ecological Engineering, 77, 242-249. Kumar, S.S., Kumar, V., Gnaneswar Gude, V., Malyan, S.K., Pugazhendhi, A., 2020. Alkalinity and salinity favor bioelectricity generation potential of Clostridium, Tetrathiobacter and Desulfovibrio consortium in Microbial Fuel Cells (MFC) treating sulfate-laden wastewater. Bioresource Technology, 306, 123110. Lee, S.J., McCormick, M.S., Lippard, S.J., Cho, U., 2013. Control of substrate access to the active site in methane monooxygenase. Nature, 494, 380-384. Li, H., Song, H., Yang, X., Zhang, S., Yang, Y., Zhang, L., Xu, H., Wang, Y., 2018. A continuous flow MFC-CW coupled with a biofilm electrode reactor to simultaneously attenuate sulfamethoxazole and its corresponding resistance genes. Science of the Total Environment, 637-638, 295-305. Li, S., Hu, Z., Zhang, J., 2017. Management and high-value utilization of constructed wetland plants: an overview. Environmental pollution and prevention, 39, 432-438. Li, W., Quan, X., Chen, L., Zheng, Y., 2020. Application of slow-release carbon sources embedded in polymer for stable and extended power generation in microbial fuel cells. Chemosphere, 244, 125515. Liu, W., Xiao, H., Ma, H., Li, Y., Adyel, T.M., Zhai, J., 2020. Reduction of methane emissions from manganese-rich constructed wetlands: Role of manganese-dependent anaerobic methane oxidation. Chemical Engineering Journal, 387. Lu, L., Xing, D., Ren, Z.J., 2015. Microbial community structure accompanied with electricity production in a constructed wetland plant microbial fuel cell. Bioresource Technology, 195, 115-121. Maltais-Landry, G., Maranger, R., Brisson, J., Chazarenc, F., 2009. Greenhouse gas production and efficiency of planted and artificially aerated constructed wetlands. Environmental Pollution, 157, 748-754. Mander, Ü., Dotro, G., Ebie, Y., Towprayoon, S., Chiemchaisri, C., Nogueira, S.F., Jamsranjav, B., Kasak, K., Truu, J., Tournebize, J., 2014. Greenhouse gas emission in constructed wetlands for wastewater treatment: A review. Ecological Engineering, 66, 19-35. Mander, Ü., Dotro, G., Ebie, Y., Towprayoon, S., Chiemchaisri, C., Nogueira, S.F., Jamsranjav, B., Kasak, K., Truu, J., Tournebize, J., Mitsch, W.J., 2014. Greenhouse gas emission in constructed wetlands for wastewater treatment: A review. Ecological Engineering, 66, 19-35. María E, H., Michelli, G., Juan Carlos, H., 2017. Greenhouse gas emissions and pollutant removal in treatmentwetlands with ornamental plants under subtropical conditions. Ecological Engineering, 35, 164-152. Meng, L., Li, X., Wang, S., Liu, L., Ma, K., Zhang, J., 2017. The long-term impact of cefalexin on organic substrate degradation and microbial community structure in EGSB system. Chemosphere, 184, 215223. Nie, W., Xie, G., Ding, J., Lu, Y., Liu, B., Xing, D., Wang, Q., Han, H., Yuan, Z., Ren, N., 2019. High performance nitrogen removal through integrating denitrifying anaerobic methane oxidation and Anammox: from enrichment to application. Environment International, 132, 105107. Oon, Y., Ong, S., Ho, L., Wong, Y., Dahalan, F.A., Oon, Y., Lehl, H.K., Thung, W., 2016. Synergistic effect of up-flow constructed wetland and microbial fuel cell for simultaneous wastewater treatment and energy recovery. Bioresource Technology, 203, 190-197. Rout, S., Parwaiz, S., Nayak, A.K., Varanasi, J.L., Pradhan, D., Das, D., 2020. Improved bioelectricity generation of air-cathode microbial fuel cell using sodium hexahydroxostannate as cathode catalyst. Journal of Power Sources, 450, 227679. Semeraro, T., Giannuzzi, C., Beccarisi, L., Aretano, R., De Marco, A., Pasimeni, M.R., Zurlini, G., Petrosillo, I., 2015. A constructed treatment wetland as an opportunity to enhance biodiversity and ecosystem services. Ecological Engineering, 82, 517-526. Song, Y., Wang, F., Liu, G., Luo, S., Guo, R., 2017. Controllable methane hydrate formation through trace carbon dioxide charging. Fuel, 203, 145-151. Srivastava, P., Abbassi, R., Yadav, A.K., Garaniya, V., Asadnia, M., 2020. A review on the contribution of electron flow in electroactive wetlands: Electricity generation and enhanced wastewater treatment. Chemosphere, 254, 126926. Srivastava, P., Yadav, A.K., Garaniya, V., Abbassi, R., 2019. Chapter 6.3 - Constructed Wetland Coupled Microbial Fuel Cell Technology: Development and Potential Applications. in: Mohan, S.V., Varjani, S., Pandey, A. (Eds.), Microbial Electrochemical Technology. Elsevier, pp. 1021-1036. Su, J., Hu, C., Yan, X., Jin, Y., Chen, Z., Guan, Q., Wang, Y., Zhong, D., Jansson, C., Wang, F., Schnürer, A., Sun, C., 2015. Expression of barley SUSIBA2 transcription factor yields high-starch low-methane rice. Nature, 523, 602-606. Tang, C., Zhao, Y., Kang, C., Yang, Y., Morgan, D., Xu, L., 2019. Towards concurrent pollutants removal and high energy harvesting in a pilot-scale CW-MFC: Insight into the cathode conditions and electrodes connection. Chemical Engineering Journal, 373, 150-160. Vymazal, J., 2011. Constructed Wetlands for Wastewater Treatment: Five Decades of Experience. Environmental Science & Technology, 45, 61-69. Wang, J., Song, X., Wang, Y., Abayneh, B., Ding, Y., Yan, D., Bai, J., 2016. Microbial community structure of different electrode materials in constructed wetland incorporating microbial fuel cell. Bioresource Technology, 221, 697-702. Wang, J., Song, X., Wang, Y., Abayneh, B., Li, Y., Yan, D., Bai, J., 2016. Nitrate removal and bioenergy production in constructed wetland coupled with microbial fuel cell: Establishment of electrochemically active bacteria community on anode. Bioresource Technology, 221, 358-365. Wang, J., Song, X., Wang, Y., Bai, J., Li, M., Dong, G., Lin, F., Lv, Y., Yan, D., 2017. Bioenergy generation and rhizodegradation as affected by microbial community distribution in a coupled constructed wetland-microbial fuel cell system associated with three macrophytes. Science of the Total Environment, 607-608, 53-62. Wang, J., Song, X., Wang, Y., Zhao, Z., Wang, B., Yan, D., 2017. Effects of electrode material and substrate concentration on the bioenergy output and wastewater treatment in air-cathode microbial fuel cell integrating with constructed wetland. Ecological Engineering, 99, 191-198. Wang, X., Daigger, G., Lee, D., Liu, J., Ren, N., Qu, J., Liu, G., Butler, D., 2018. Evolving wastewater infrastructure paradigm to enhance harmony with nature. Science Advances, 4. Weaver, A.J., 2011. Toward the Second Commitment Period of the Kyoto Protocol. Science 332 (6031), 795-796. Wetser, K., Sudirjo, E., Buisman, C.J.N., Strik, D.P.B.T., 2015. Electricity generation by a plant microbial fuel cell with an integrated oxygen reducing biocathode. Applied Energy, 137, 151-157. Xiao, N., Wu, R., Huang, J.J., Selvaganapathy, P.R., 2020. Anode surface modification regulates biofilm community population and the performance of micro-MFC based biochemical oxygen demand sensor. Chemical Engineering Science, 221, 115691. Xu, L., Zhao, Y., Tang, C., Doherty, L., 2018. Influence of glass wool as separator on bioelectricity generation in a constructed wetland-microbial fuel cell. Journal of Environmental Management, 207, 116-123. Yadav, A.K., Dash, P., Mohanty, A., Abbassi, R., Mishra, B.K., 2012. Performance assessment of innovative constructed wetland-microbial fuel cell for electricity production and dye removal. Ecological Engineering, 47, 126-131. Yang, H., Bao, B., Liu, J., Qin, Y., Wang, Y., Su, K., Han, J., Mu, Y., 2018. Temperature dependence of bioelectrochemical CO2 conversion and methane production with a mixed-culture biocathode. Bioelectrochemistry, 119, 180-188. Yousefi, V., Mohebbi-Kalhori, D., Samimi, A., 2017. Ceramic-based microbial fuel cells (MFCs): A review. International Journal of Hydrogen Energy, 42, 1672-1690. Zhang, K., Liu, Y., Chen, Q., Luo, H., Zhu, Z., Chen, W., Chen, J., Mo, Y., 2018. Effect of submerged plant species on CH4 flux and methanogenic community dynamics in a full-scale constructed wetland. Ecological Engineering, 115, 96-104. Zhang, K., Wu, X., Luo, H., Li, X., Chen, W., Chen, J., Mo, Y., Wang, W., 2020. CH4 control and associated microbial process from constructed wetland (CW) by microbial fuel cells (MFC). Journal of Environmental Management, 260, 110071. Zhang, S., You, J., Chen, H., Ye, J., Cheng, Z., Chen, J., 2020. Gaseous toluene, ethylbenzene, and xylene mixture removal in a microbial fuel cell: Performance, biofilm characteristics, and mechanisms. Chemical Engineering Journal, 386, 123916. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-202429","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":11580177,"identity":"93b11390-c7ae-4e4e-8af5-2994fe7d8db1","order_by":0,"name":"Ke Zhang","email":"","orcid":"","institution":"Harbin institute of technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Zhang","suffix":""},{"id":11580178,"identity":"96b32371-4088-4127-8ae6-e5424a534afa","order_by":1,"name":"Xiangling Wu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiangling","middleName":"","lastName":"Wu","suffix":""},{"id":11580179,"identity":"91ae08ea-2785-44bf-98d5-4a4f73261f75","order_by":2,"name":"Wei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACA2YwJQHj2/Dw8zcQ0sKMrCUhTUZyxgECWhiYkbkJh20MGhLwazFn5z/4uLDNIp9fujvxceGP8zwGDAcYP3zMwa3FspmZ2Xhmm4TlzDlnNxvPSLjNY87cwCw5cxsehx1mZpPmOSNhYHAjd5s0D1CLZcMBNmZe/FrYf0O1bP/Nk3COx+BAAkEtbMw8FRBbmHkSDhClxVgapEVyRu5maZ60ZB7JGQeb8fvl/MGHn3kM6gz4JXI3fuaxsbPn528++OEjHi3YAGMDaepHwSgYBaNgFGAAAK6RSAK3gQRdAAAAAElFTkSuQmCC","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":11580180,"identity":"e8d9294e-a5e2-4e5e-b35b-26e1487d59f2","order_by":3,"name":"Hongbing Luo","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongbing","middleName":"","lastName":"Luo","suffix":""},{"id":11580181,"identity":"6c30b967-fc26-4a30-8014-771234563ab0","order_by":4,"name":"Wei Chen","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Chen","suffix":""},{"id":11580182,"identity":"7d80961f-4177-4922-a6ba-00b7eb8c6119","order_by":5,"name":"Jia Chen","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2021-02-03 23:00:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-202429/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-202429/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6074086,"identity":"0df3ef16-a76f-4bb2-b73c-6470e8dedbf6","added_by":"auto","created_at":"2021-02-17 22:49:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136753,"visible":true,"origin":"","legend":"Schematic diagram of different types of CW-MFCs.","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/de7b1111fd9fc529548428c6.png"},{"id":6073745,"identity":"442417cf-f261-415c-ad5d-140067beadb6","added_by":"auto","created_at":"2021-02-17 22:46:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78864,"visible":true,"origin":"","legend":"The average CH4 fluxes from different CW-MFCs under different hydraulic retention time HRT (a), Box plots of CH4 flux from different CW-MFCs (b). Values are mean ± standard deviation. Error bars are ± 1 standard deviation from the test mean (n=3).","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/889d618e59a8a830bf988a4c.png"},{"id":6074087,"identity":"786d74b1-7d3c-4293-aec9-d366006c85c4","added_by":"auto","created_at":"2021-02-17 22:49:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99210,"visible":true,"origin":"","legend":"COD removal rates in different MFC-CWs (open-circuit and close circuit conditions) under different HRT. Error bars are ± 1 standard deviation from the test mean (n=3).","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/c85f236aebf7729aac02b1e8.png"},{"id":6074448,"identity":"1fba3718-0bae-4a39-afc2-8ea928a68182","added_by":"auto","created_at":"2021-02-17 22:52:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60511,"visible":true,"origin":"","legend":"Voltage values of four CW-MFCs under closed - circuit conditions.","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/6c6551f86eba306aed6f3935.png"},{"id":6074447,"identity":"344cd337-2235-4077-aa55-c994ad186d81","added_by":"auto","created_at":"2021-02-17 22:52:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":93992,"visible":true,"origin":"","legend":"Power density and polarization curves of four CW-MFCs under closed- circuit conditions (the external resistance ranging from 50Ω to 90 kΩ). ( a) p1-ca, (b)p1-an, (c)p2-ca, (d)p2-an.","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/0a503f91a296eecf44338c0b.png"},{"id":6074090,"identity":"936c85b1-89c3-4264-b1b4-613f93f21f78","added_by":"auto","created_at":"2021-02-17 22:49:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":71980,"visible":true,"origin":"","legend":"Quantification of methanogens and methanotrophs based on mcrA and pmoA genes by q-PCR. Error bars are ± 1 standard deviation from the test mean (n=3).","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/f86bebb2e8b83a42ca6a8833.png"},{"id":6074091,"identity":"8178c199-9df2-4e9c-966d-a66678a36fc0","added_by":"auto","created_at":"2021-02-17 22:49:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":154795,"visible":true,"origin":"","legend":"Bacterial community compositions in different CW-MFCs based on 16s gene analysis. (a) at the phylum level, (b) at the genus level.","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/b7cca91d0c38066fd38b90fd.png"},{"id":6073753,"identity":"4872ccee-2806-4a3e-bfaf-e381eae0565a","added_by":"auto","created_at":"2021-02-17 22:46:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":81099,"visible":true,"origin":"","legend":"Methanogenic archaea community compositions from different CW-MFCs based on Illumina Miseq sequencing.","description":"","filename":"OnlineFig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/285052035086a33d9bfc479e.png"},{"id":6073749,"identity":"9fdaf313-b7aa-440e-a0ae-f781dbeaec06","added_by":"auto","created_at":"2021-02-17 22:46:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":191986,"visible":true,"origin":"","legend":"Scanning electron microscope (SEM) micrographs of bioanode from CW-MFCs. (a) P1-Ca ×20 k, (b) P1-An ×50 k, (c)P2-Ca, ×20 k, (d) P2-An ×50 k. ","description":"","filename":"OnlineFig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/8f14f936adbea809696cadfe.png"},{"id":13666311,"identity":"156c353f-f523-4241-872f-d6d226cd8e34","added_by":"auto","created_at":"2021-09-17 10:48:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1914085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-202429/v1/e9aec69d-c6de-4183-9a2c-f4dc4517a1b7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eApplication of Bioelectrochemical Technique in Energy Recovery and CH4 Control From Constructed Wetland (CW) and Associated Biochemical Mechanisms\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":" \u003cp\u003eIn the context of global warming and the increasingly significant impact of human activities on the environment, greenhouse gas emission reduction, low-carbon economy and low-carbon cities are the research hotspots nowadays \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Global climate change caused by the increase of greenhouse gas emissions in the atmosphere has become the focus of human concern, and is also a huge challenge facing the sustainable development of the world economy. Methane is an important greenhouse gas, which is considered to be one of the most important greenhouse gases after CO\u003csub\u003e2\u003c/sub\u003e. Its greenhouse effect is 20\u0026ndash;30 times that of CO\u003csub\u003e2\u003c/sub\u003e \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, which contributes about 20% of the global climate directly drive \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Methane has become an important part of global \"carbon reduction\".\u003c/p\u003e \u003cp\u003eConstructed wetland (CW) is considered as a reliable wastewater treatment technology, which has the advantages of less investment, less operation cost, easy maintenance and stable effluent quality\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, which plays an important role in non-point source pollution control. However, constructed wetlands will release greenhouse gases such as CO\u003csub\u003e2\u003c/sub\u003e, CH\u003csub\u003e4\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO during operation, which will have a negative impact on the environment. Studies showed that constructed wetlands emit 2 to 10 times more greenhouse gases than natural wetlands \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, its role in global warming cannot be ignored. In recent years, scholars have also carried out research on methane emission reduction in CWs. In general, methane emission control in CWs can be divided into two categories: first, macro control, including physical control methods such as aeration\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, Chemical methods (e.g. REDOX potential control, iron ion control, chemical substance control)\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, Hydrologic method (influent pattern and water level variation) etc \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Another method is micro-control, such as molecular biological methods \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e and nano-control \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. So far, however, study on the microscopic control of methane emissions is limited. In particular, the literature on methane control by microbial or bioelectrochemical method is quite limited.\u003c/p\u003e \u003cp\u003eMicrobial fuel cell (MFC) is a device that using electrogenic microorganisms to directly convert chemical energy of organic matter into electrical energy. In the past 10 years, a lot of work has been done to improve the electrical performance of MFC, wastewater treatment efficiency, reactor configuration and electrode materials \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. But it is a pity that its electricity generation efficiency is still very low and its engineering application is limited. Therefore, the development and application of MFC in the academic community caused controversy and doubt \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In fact, we can change our thinking and use MFC as a tool rather than as a research purpose. Due to the similar operating conditions of microbial fuel cell and constructed wetland, the integration of MFC with CW technology becomes possible. Since CW-MFC integrated technology was first proposed in 2012\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, it has been proved in recent years that CW-MFC Technology can greatly improve the sewage treatment efficiency and electricity generation performance\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Theoretically, there exists a competitive relationship between electrogenic microorganisms and methanogens in the anodes of MFC and CW, which can inhibit methane emission. Our previous study confirmed that methane emission could be inhibited by running MFC \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. However, the research on the regulation of methane emission in CW by bioelectrochemical method (MFC) is still inadequate and very limited. In addition, there are still many scientific problems to be further explored. It is not clear, for example, whether the type of plant and the location of plant root system on the electrode has an effect on methane emissions, how much, and how it works. In the current CW-MFCs studies, the plant roots were mostly placed at the anode\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, while in some studies, the plant roots were mostly placed at the cathode. Theoretically, placing them at the anode would provide organic matter for electricity generation, while placing them at the cathode would increase the number of electron receptors needed to generate electricity. However, it has not been reported whether the plant roots placed at the anode or the cathode are more conducive to electricity generation and methane emission.\u003c/p\u003e \u003cp\u003eTherefore, this study aims to further explore the influence mechanism of MFC on CW methane emissions, wastewater treatment and electricity generation based on the above problems. The influence of the electrode position of plant root system on methane emission and electrical performance and associated biochemical processes were mainly explored. The mechanism underlying competition between electrogenesis and methanogenesis at the anode was investigated. The systematic elucidate of the biological process involved in the integrated system (CW-MFC) will provide a theoretical basis for CH\u003csub\u003e4\u003c/sub\u003e control in constructed wetlands and promote the sustainable development of wastewater treatment.\u003c/p\u003e "},{"header":"2. Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1. MFC-CW construction and operation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight polypropylene plastic circular columns with the height of 0.55 m and the diameter of 0.18 cm were adopted as the main reactors to simulate the vertical flow CW-MFC system. Two plant species (\u003cem\u003eTypha orientalis, Cyperus alternifolius\u003c/em\u003e) were planted in anodes and cathodes, respectively. In CW-MFCs planted with\u003cem\u003e Typha Orientalis\u003c/em\u003e, plant roots located at the cathode and anode were named as P1-Ca and P1-An, respectively. In CW-MFCs planted with\u003cem\u003e Cyperus alternifolius\u003c/em\u003e), plant roots located at the cathode and anode were named as P2-Ca and P2-An, respectively. The other four reactors operated under open circuit as a control, marked as CK1, CK2, CK3 and CK4. From the bottom to the top of the reactor are: the first layer is 10 cm thick gravel, the second layer is 10 cm thick granular activated carbon GAC (anode layer), the third layer is 20 cm thick gravel, and the fourth layer is 5 cm thick granular activated carbon (cathode layer). Stainless steel wire mesh (12 cm in diameter) is placed in the middle of the anode layer and the cathode layer respectively to collect and transfer electrons. The cathode and anode are connected by titanium wire, and the external copper wire is connected with the resistance box to form a closed loop. The schematic diagram of the CW-MFCs is shown in Fig.1, the control reactors in open circuit are not shown. In this study, the selection of electrode materials, the setting of electrode distance and the configuration were according to the reported literature \u003csup\u003e[4]\u003c/sup\u003e , which has good power generation, wastewater treatment capacity and economic feasibility.\u003c/p\u003e\n\u003cp\u003eActivated sludge was taken from a local wastewater treatment plant. The aerobic activated sludge was inoculated at the cathode of CW-MFCs and the anaerobic activated sludge was inoculated at the anode of CW-MFCs. The reactor operated in a continuous flow mode with water inflow at the bottom. The synthetic wastewater was composed as follows: KCl 130.0 mg/L, Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO 275.0 mg/L, NH\u003csub\u003e4\u003c/sub\u003eCl 133.7 mg/L, NaHCO\u003csub\u003e3\u003c/sub\u003e 313.0, MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 25.0 mg/L, CaCl\u003csub\u003e2\u003c/sub\u003e 15.0 mg/L, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO 497.0 mg/L, trace elements 0.1 ml/L. The influent COD was controlled at 300 mg/L. The hydraulic residence time was set according to the experimental requirement. The reactors were operated at 26 \u0026plusmn; 2 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2 COD measurement and CH\u003csub\u003e4\u003c/sub\u003e fluxes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemical oxygen demand (COD) was determined by potassium dichromate method. The methane was collected by a static chamber method and analyzed by a gas chromatograph (PerkinElmer GC Clarus 680). The injection temperature was 85\u0026deg;C, the split ratio was 20:1, the column temperature was 75 \u0026deg;C, and the gasification temperature was 250 \u0026deg;C. The content of CH\u003csub\u003e4\u003c/sub\u003e was calculated by the linear relationship between the peak area and the known content. The methane emission flux was calculated by the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" alt=\"\" /\u003e\u003c/p\u003e\n\u003cp\u003eIn the equation: E\u003csub\u003emethane\u003c/sub\u003e -- methane emission flux, g/m\u003csup\u003e2\u003c/sup\u003e/h;\u0026nbsp;\u0026rho;--The density of methane in standard condition, 714.3 g/m\u003csup\u003e3\u003c/sup\u003e; V -- Closed volume of collected gas, m\u003csup\u003e3\u003c/sup\u003e; A -- Area of electrode surface, m\u003csup\u003e2\u003c/sup\u003e; dc methane/dt -- Change in methane gas in a given time, mg/ mL /h; T -- average temperature\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3 Bioelectricity generation monitoring\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioelectricity generation performance was measured under the resistance of 1000 \u0026Omega; by data acquisition system (model 2700, Keithley Inc.). The voltage data was recorded every 5 minutes. The current value is obtained according to voltage and resistance (I=U/R). Subsequently, power density (P, W/m\u003csup\u003e3\u003c/sup\u003e) and current density (J, A/m\u003csup\u003e3\u003c/sup\u003e) were calculated according to the anode volume. The current density and power density were calculated as J = U/RA and P = IU/A, respectively. Where, I is the current, U is the voltage, R is the resistance and A is the working volume of the anode. The polarization curve was determined by changing the external electrical resistance from 50 to 90,000 \u0026Omega; at the end of experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.4 DNA extraction and Illumina sequencing \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total DNA of samples was extracted by a Power Soil DNA Isolation Kit 178 (MOBIO Laboratories Inc., USA) according to the manufacturer\u0026rsquo;s instructions. Bacterial 16S rDNA and archael gene for pyrosequencing were amplified by PCR. The primers BAC27F (5'-AGAGTTTGATCCTGGCTCAG-3'), BAC1492R (5'-GGTTACCTTGTTACGACTT-3' and the primers ARC25F (5'-CYGGTTGATCCTGCCRG-3'), ARC958R (5'-YCCGGCGTTGAMTCCAATT-3') were used for bacterial and archaea, respectively. The products of PCR were loaded on 1.5% agarose gel and the gel was purified with Gel Extraction Kit (OMEGA Bio-tekInc, USA). The DNA concentration and quality were determined by real-time PCR (FTC-3000\u0026trade;), subsequently the amplicons were further used for Illumina MiSeq sequencing. All the raw reads were analyzed by combining different programs from the quantitative insights into microbial ecology (QIIME) after sequencing. When the low quality adaptors, primers, barcodesand reads were eliminated, the remaining 16S rRNA sequences were clustered into OTUs using UCLUST with the distance of 0.03.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5 Quantitative PCR ( q-PCR) of functional genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe function genes for methanogens and methane-oxidizing bacteria were determined by q-PCR on an ABI 7500 real-time PCR system instrument (Applied Biosystems, USA). Primers mcrA-f (5\u0026prime;-GGTGGTGTMGGATTCACACARTAYGCWACAGC-3\u0026prime;) and \u003cem\u003emcr\u003c/em\u003eA-r (5\u0026prime;-TTCATT GCRTAGTTWGGRTAGTT-3\u0026prime;) were used for \u003cem\u003emcr\u003c/em\u003eA gene amplification. Primers A189-f (5\u0026prime;-GGNGACTGG GACTTCTGG-3\u0026prime;) and mb661-r (5\u0026prime;-CCGGMGCAACGTCYTTACC-3\u0026prime;) were used for \u003cem\u003epmo\u003c/em\u003eA gene amplification. The reaction volume, the reaction temperature, the reaction procedure of PCR amplification and the plasmid curve were plotted according to our previously reported methods\u003csup\u003e[42]\u003c/sup\u003e .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.6 Scanning electron microscope (SEM) analysis of bioanode\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of anode microorganism was imaged using SEM (JSM-5610, Japan). The samples were treated as follows: A small amount of sample was placed in a 5 mL centrifuge tube, 2.5% glutaraldehyde was added, and fixed in the refrigerator for 1.5 hours. And then rinsing with phosphate buffer solution of 0.1mol/L (pH=6.8) for three times. The samples were dehydrated with 50%, 70%, 80% and 90% ethanol in turn. Rinse with a mixture of anhydrous ethanol and isoamyl acetate at volume ratio of 1:1 for 15 minutes, then rinse with pure isoamyl acetate for 15 minutes. The dried sample was pasted on the sample table of SEM and coated with Au film of 1500 nm thickness on the sample surface. Finally, the samples were observed under SEM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7\u003cem\u003e Statistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using SPSS software (version 21.0) (SPSS Inc., Chicago, USA). Significance analysis was used to identify differences between experimental groups or control groups by one-way ANOVA at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.The R software package and Origin (version 8.0) were used for bioinformatic analysis graphical works.\u003c/p\u003e"},{"header":"3. Results And Discussion","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 CH\u003csub\u003e4\u003c/sub\u003e emissions from CW-MFCs\u003c/h2\u003e \u003cp\u003eAfter four weeks of batch operation, the voltage in all reactors reached a steady state. The reactors then operated in a continuous flow mode and began recording data. During the experiment, the plant death in two reactors led to abnormal data (CK3, CK4), so the data of these two reactors would not be recorded in the subsequent experiment. So there are six reactors running stably. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, CH\u003csub\u003e4\u003c/sub\u003e emissions from all reactors showed a downward trend with the increase of hydraulic residence time (HRT). CH\u003csub\u003e4\u003c/sub\u003e emission was significantly higher in open circuit than in closed circuit. When HRT\u0026thinsp;=\u0026thinsp;1, the average CH\u003csub\u003e4\u003c/sub\u003e emissions of CK1 and CK2 reached 6.1 mg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 6.4 mg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), indicating that MFC operation in constructed wetlands can effectively reduce methane emission. When plant roots were located at the anode, methane emission was significantly higher than that at the cathode (P1-an =\u0026thinsp;3.9 mg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, P2-an =\u0026thinsp;4.5 mg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). CH\u003csub\u003e4\u003c/sub\u003e emissions was lowest when plant roots were located at the cathode, with an average of 2.3 mg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e found in P1-Ca. Statistical analysis showed that both plant and root location had an impact on methane emission, but the impact of plant root location on methane emission was more significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). CH\u003csub\u003e4\u003c/sub\u003e emissions from the reactor planted with \u003cem\u003eTypha Orientalis\u003c/em\u003e were lower than those of the reactor planted with \u003cem\u003eCyperus Alternifolius\u003c/em\u003e both at the cathode and anode.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith the increase of HRT, methane emission in CW-MFC tends to decrease, which is consistent with previous research conclusion of CH\u003csub\u003e4\u003c/sub\u003e emissions in CW \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Although a lot of work has been done on the role of plants in CWs, consensus has been reached in the academic circle that plants have little impact on the wastewater treatment efficiency of constructed wetlands. The contribution of plants to sewage treatment is about 10%, while the fillers, matrix, operation modes and other factors contribute 90% of sewage treatment efficiency \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. However, different plant species on the bioelectricity generation and CH\u003csub\u003e4\u003c/sub\u003e emissions in CW-MFCs have not been fully explored. From our results, it can be seen that plants played an important role in CH\u003csub\u003e4\u003c/sub\u003e in CW-MFCS. Therefore, from the perspective of sustainable development of wastewater treatment, more attention should be paid to plant species selection during CW development and management.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 COD removal efficiency\u003c/h2\u003e \u003cp\u003eThe removal efficiency of COD was measured under different HRT. The results show that the removal efficiency of COD increases with the increase of HRT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Except for P1-An, the COD removal efficiency was positively correlated with HRT. The removal rates of COD in close-circuit reactors were significantly higher than that of open-circuit reactors. This result indicated that the operation of MFC effectively promoted the degradation of organic matter. The average COD removal rate was about 85% in the control groups. In contrast, the average COD removal rate is more than 90% in the CW-MFC reactors. Previous studies have also shown that MFC integrated with CW could significantly promote the efficiency of sewage treatment\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. The probable reason may be that electrochemical processes can promote the efficiency of electron transfer, thus effectively improving the degradation of pollutants\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. When plant roots were located at the cathode, COD removal efficiency was higher than that of plant roots at the anode. In the P1-Ca reactor, the average COD removal rate reached 95.1%. Unlike the removal of COD, there was no significant difference between two plant species (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).This results were consistent with previous studies on the effectiveness of plants in removing pollutants from CWs \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Bioelectricity performance\u003c/h2\u003e \u003cp\u003eThe bioelectricity generation performances in four CW-MFCs were monitored. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the average voltage generated by the four reactors varied significantly. The voltage of plant roots at the cathode was greater than that at the anode (P1-Ca\u0026thinsp;=\u0026thinsp;490 mV, P1-An =\u0026thinsp;437 mV). P2-Ca\u0026thinsp;=\u0026thinsp;354 mV, P2-An =\u0026thinsp;321 mV). Different from the influence of plants on COD removal efficiency, plant species had a significant influence on voltage. The voltage generated by the reactor growing \u003cem\u003eTypha Orientalis\u003c/em\u003e was higher than that generated by the reactor planted \u003cem\u003eCyperus Alternifolius\u003c/em\u003e, whether the plant roots were at the anode or the cathode. This may be related to the type and amount of exudates and organic matter in the root system of different plant species\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The organics from plant rhizodeposits can provide more electron donors for the electrochemical active bacteria (EAB). Plant roots at the cathode have a better electrical performance than plants at the anode, probably because they have limited electron receptors in the system, and the oxygen produced by plants increases the electron receptors\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Thus, the electrical performance of the reactor is improved. However, the effect of the improved electron acceptor brought by the plant root at the cathode on the electric efficiency was less than that of the plant species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe polarization curve and the power density curve of four CW-MFCs were obtained by changing the external resistance from 50 Ω to 90 kΩ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The P1-Ca produced maximum power density of 0.22 W m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. The corresponding current density was 0.4 A m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. The lowest power density was observed in P2-An with 0.058 W m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. In the reactor planted with \u003cem\u003eCyperus Alternifolius\u003c/em\u003e, the power density was less than 0.14 W m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. On the contrary, in the reactor where \u003cem\u003eTypha Orientalis\u003c/em\u003e was planted, the power density was above 0.22 W m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. These results indicated that plant species had a significant effect on power density in CW-MFC systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to the enhanced performance of wastewater treatment and bioelectricity generation, bioelectrochemical systems (MFC and MEC) integrated constructed wetlands have gained a considerable amount of attention\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The enhancement of wastewater treatment and bioelectricity generation mainly emanated from the electron transfer or flow, particularly in anaerobic areas\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. However, there are few studies on the influence of plant roots location on the electrical performance in CW-MFCs system, and the references available are still very limited. Previous study showed that plant roots located at the anode could improve the electrical performance due to root exudates and other reasons\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In our study, it is found that when plant roots were located at the cathode, the role of plant in increasing electron donor is greater. The existence of electron donors is conducive to improving the electrical performance\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. However, at present, both CW and CW-MFC have low power generation, which is difficult to be applied in engineering. Inadequate electron transfer may be one of the important reasons for the low efficiency of electricity generation. Therefore, further understanding the electron transfer between electrode-anode and cathode in CW-MFCs is essential to improve the development of this integrated technology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 q-PCR of mcrA genes and pmoA genes\u003c/h2\u003e \u003cp\u003eTo quantify the methanogens and methanotrophs in reactors, samples were collected from the anode and cathode for the determination of these two bacteria, respectively. The quantification of \u003cem\u003emcrA genes and pmoA\u003c/em\u003e genes, which encode methanogenesis and methanoxase, were investigated by q-PCR. The results showed that the number of \u003cem\u003emcrA genes\u003c/em\u003e in open-circuit systems was greater than that in closed-circuit systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The number of \u003cem\u003emcrA\u003c/em\u003e genes at the anode was greater than that of cathode. Meanwhile, the differences between plant species were observed. In the reactor planted with \u003cem\u003eCyperus Alternifolius\u003c/em\u003e, the average number of \u003cem\u003emcrA\u003c/em\u003e gene is 3.6\u0026times; 10\u003csup\u003e4\u003c/sup\u003e copies mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In contrast, in the reactor planted with \u003cem\u003eTypha Orientalis\u003c/em\u003e, the average number of \u003cem\u003emcrA genes\u003c/em\u003e is 4.7\u0026times; 10\u003csup\u003e4\u003c/sup\u003e copies mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Correlation analysis showed that there was a significant positive correlation between the number of methanogens and CH\u003csub\u003e4\u003c/sub\u003e production in the reactor \u003cem\u003e(r\u0026thinsp;=\u0026thinsp;0.98, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/em\u003e. Unlike \u003cem\u003emcrA\u003c/em\u003e genes, \u003cem\u003epmoA\u003c/em\u003e genes increased slightly in closed-circuit systems. At the same time, the number of \u003cem\u003epmoA\u003c/em\u003e genes in the plant root at the cathode was greater than that in the anode. The influence of plant species on the number of \u003cem\u003epmoA\u003c/em\u003e genes is not obvious. Correlation analysis showed there was no significant correlation between CH\u003csub\u003e4\u003c/sub\u003e emissions and the number of \u003cem\u003epmoA\u003c/em\u003e genes, which suggests that plants not only affect CH\u003csub\u003e4\u003c/sub\u003e emissions by influencing methanotrophs, but also through their physiological functions, such as plant conduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMethane emission is the net result of methane production, oxidation and transport\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The production and oxidation of methane are closely related to microorganisms, especially methanogens and methane-oxidizing bacteria (methanotrophs) are important biological factors that determine methane emission. However, from the results of this study, there was no significant correlation between CH\u003csub\u003e4\u003c/sub\u003e emissions and methanotrophs, indicating that other factors may play a key role. Plants play an important role in methane production and transport. On the one hand, the organic matter and plant rhizodeposits provided by different plants can lead to different CH\u003csub\u003e4\u003c/sub\u003e emissions. On the other hand, plants affect the redox potential in the rhizosphere zone and the ratesof oxygen transport via plant aerenchyma from the rhizosphere\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. In addition, some plants have been reported to release methane under aerobic conditions\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Therefore, a lot of systematic studies about the influence of plant species on CH\u003csub\u003e4\u003c/sub\u003e emissions in the CW-MFCs are still needed, rather than the current studies on phenomena.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Bacterial and archael community analysis\u003c/h2\u003e \u003cp\u003eThe bacterial community composition at phylum level is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea. Bacteroidetes and Planctomycetes were the predominant community in CKs, which accounted for 21% \u0026minus;\u0026thinsp;29% of total composition. In contrast, their abundances in the other MFC-CWs were low. Chloroflexi and Proteobacteria were dominant community in close circuit MFC-CWs, which was significantly higher than that of CKs. Proteobacteria as the most abundant population in P1-CA reactor accounted for 29%, followed by Chloroflexi (15%). In addition, the abundance of Firmicutes in close circuit MFC-CW was higher than that of CKs. The community abundance of other phyla did not differ significantly in all reactors. At the genus level, the community composition of all reactors also showed significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The genera of exoelectrogens (\u003cem\u003eGeobacter, Geothrix\u003c/em\u003e and \u003cem\u003eDesulfobulbus\u003c/em\u003e) were not detected in CKs. The main community composition of CKs was \u003cem\u003eSaprospiraceae\u003c/em\u003e and \u003cem\u003eArthrobacter\u003c/em\u003e. P1-Ca and P2-Ca had high abundance of exoelectrogens. The highest value (21%) of \u003cem\u003eGeobacter\u003c/em\u003e was observed in P1-Ca. All the reactors had the nitrification and denitrification community (\u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eBrachymonas\u003c/em\u003e and \u003cem\u003eNitrosomonas\u003c/em\u003e). In addition, \u003cem\u003eAnaerolinea\u003c/em\u003e was found had high percentage in close circuit MFC-CWs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA high proportion of Proteobacteria was found in the reactor, which may be related to the formation of exoelectrogens and the increase of nitrification and denitrification bacteria\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. These bacteria belong to the pylum of Proteobacteria. High abundance of Firmicutes in CC MFC-CW resulted in high electricity generation and COD removal. Firmicutes belongs to cellulolytic bacteria, which can hydrolyze cellulose derived from dead plant roots into smaller compounds like fatty acids\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Theses acids can then be utilized by EAB to electricity generation. Chloroflexi belongs to fermentative bacteria, can ferment small molecular saccharides to short-chain fatty acids and H\u003csub\u003e2\u003c/sub\u003e \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, which can be used as electron donors for EAB. Therefore, their high proportion in CC MFC-CWs promotes the increase of current in the reactor. The common dephosphorization bacteria (Accumulibacter phosphatis and Tetrasphaera) in wastewater treatment plants were not detected, which indicated that the removal of phosphorus in the MFC-CWs was mainly through the absorption of plant rather than biological removal. The denitrification bacteria (Nitrospira, Brachymonas and Nitrosomonas), which were widely detected in the municipal sewage treatment plant, were detected in the Closed Circuit MFC-CWs, indicating that they have potential for denitrification. The Geobacter was widely reported as exoelectrogen in MFC. While the Desulfobulbus and Geothrixas were rarely reported in MFC-CW. Its role remains to be further studied.\u003c/p\u003e \u003cp\u003eThe archeal community composition is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Methanosaeta and Methanosarcina were the dominant archae in both cathode and anode in CK. Methanosaeta were also predominant in all the other reactors, but their relative abundance dropped around 40% in close circuit CW-MFCs. Hydrogenotrophic methanogens (Methanobacteriales and Methanococcales) showed an increased trend in all the electrical generation reactors. Methanopyrales, Thermococcales and Desulfurococcales were not detected in CKs, but they were in high percentage in electrical generation reactors. The higher values of Methanosarcina were observed in P1-Ca and P2-Ca, with 20% and 15% abundance, respectively. At the same time, about 8% of the archaea were not identified,suggesting a large number of unknown archaea.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt present, there is very limited information about archaea communities and their roles in CW-MFCs. The decrease of strictly acetotrophic methanogens (Methanosaetaceae) in close circuit reactors was related the hydrogen ions formation and competition between acetotrophic archaea and bacteria\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. On contrary, the hydrogenotrophic methanogens (Methanobacteriales and Methanococcales) tended to increase, the probable reason was that the formation of hydrogen ions and electrons during electricity generation improved the activity of hydrogenotrophic methanogens \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Thermococcales and Desulfurococcales are the hyperthermophilic archaea, which were seldom reported in previous studies regarding the archeal community of MFCs. Thermococcales and Desulfurococcales were reported only in plant fuel cells \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, their role is unclear. Therefore, the role and mechanism of these hyperthermophilic archaea need to be further studied. Methanopyrales has not been reported in archaeal communities of MFCs. Desulfurococcales were reported to be autotrophic and heterotrophic. Some of them are autotrophic archae using hydrogen as the electron donor and sulfur, nitrate, nitrite and thiosulfate as the electron acceptor, some of them can also grow through organic fermentation \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. They were predominant in P1-Ca and P2-Ca, resulting in high electricity generation. They may participate in fermentation process to produce simple material for electricity generation and methane inhibition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 SEM analysis of electrode microorganisms\u003c/h2\u003e \u003cp\u003eThe morphologies of the electrode biofilm collected from reactors were scanned using SEM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, various forms of microorganisms, including cocci and bacilli, were adsorbed on the electrode surface. In particular, in P1-Ca reactors, numerous types of microbes were found to be tightly attached to the electrodes including spiral, rod and spherical. Microbes on the anode are more diverse than those on the cathode. At present, the morphology of anode microorganisms has been studied intensively, but the morphology and function of cathode microorganisms have been studied less. Nanowires, circled in red, were also found on the microbial surface except P2-An. These wires originated from the cell surface and extend in different directions. Interspecific electron transport, including direct electron transport (DIET) and indirect electron transport, greatly accelerates biochemical reactions\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. There are three types of interspecific direct electron transport: REDOX proteins, nanowires, and electron shutters (riboflavin, anthraquinone). One of the most common is nanowires. The number and mode of nanowires have an important impact on the efficiency of electricity generation\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. In this study, the largest number of nanowires was found in the P1-Ca reactor, which may also be related to the high efficiency of electricity generation. How to enhance the interspecific electron transfer and overcome the electrolyte resistance and charge transfer resistance caused by too slow reaction rate on the electrode are the keys to improve the efficiency of CW-MFC. Therefore, more relevant studies are needed in the future.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, we mainly focused on the effects of plants and their roots location on CH4 emission, electricity generation and COD removal, and did not focus on the effects on electrode materials and electrode distance. In fact, electrode materials and electrode distance may have a great influence on the performance of CW-MFCs\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e,\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. At present, there are few studies on the relationship between the configuration and performance of CW-MFC reactor. The effect of reactor configuration on CH\u003csub\u003e4\u003c/sub\u003e emissions has not been studied. In addition, the current research on CW-MFC is in the experimental scale, and the actual scale of the system is still in the development stage.\u003c/p\u003e \u003c/div\u003e "},{"header":"4. Conclusion","content":"\u003cp\u003eThe results showed that the operation of MFC could not only improve the wastewater treatment efficiency of CW, but also effectively reduce the methane emission, mainly through the competition between electrochemical active bacteria (EAB) and methanogenes at the anode. EAB were more advantageous than methanogens in utility of carbon sources and became the dominant bacteria group of anode. Plant roots located at the cathode are more conducive to electricity generation and CH\u003csub\u003e4\u003c/sub\u003e control. The enhancement effect of plant roots as electron donors in CW-MFC system was greater than that of root exudates as electron donors. At the same time, plants species played a more important role in CH\u003csub\u003e4\u003c/sub\u003e emissions in the CW-MFC system than the location of plant roots at the electrode. Although our study showed that running MFC in CW and plant roots located at the cathode were more conducive to energy recovery and CH\u003csub\u003e4\u003c/sub\u003e control, its durability of CH\u003csub\u003e4\u003c/sub\u003e emissions mitigation over a long period of time is still a challenge.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFundings \u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (No. 51808363) and Foundation of Chengdu Science and Technology Bureau (No. 2019-YF05-00839-SN)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions Ke Zhang:\u003c/strong\u003e\u0026nbsp;Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eXiangling Wu:\u003c/strong\u003e\u0026nbsp;Methodology, Writing - review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eWei Wang:\u003c/strong\u003e\u0026nbsp;Methodology, Writing - review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eHongbing Luo:\u003c/strong\u003e\u0026nbsp;Writing - review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eWei Chen:\u003c/strong\u003e\u0026nbsp;Formal analysis, Writing - review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eWei Chen:\u003c/strong\u003e\u0026nbsp;Data curation, Formal analysis, Writing - review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eJia Chen:\u003c/strong\u003e\u0026nbsp;Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval \u003c/strong\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate \u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish \u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBruhn, D., M\u0026oslash;ller, I.M., Mikkelsen, T.N., Ambus, P., 2012. Terrestrial plant methane production and emission. Physiol Plantarum. 12, 117-124.\u003c/li\u003e\n\u003cli\u003eCarlos, R., Amanda, P., Carlos, A., Pedro, C., Abraham, E.E., 2018. Microbial electrochemical technologies for wastewater treatment: principles and evolution from microbial fuel cells to bioelectrochemical-based constructed Wetlands. Water Res. 10(9), 1128-1135.\u003c/li\u003e\n\u003cli\u003eFan, L., Dippold, M.A., Ge, T., Wu, J., Thiel, V., Kuzyakov, Y., Dorodnikov, M., 2020. Anaerobic oxidation of methane in paddy soil: Role of electron acceptors and fertilization in mitigating CH4 fluxes. Soil Biology and Biochemistry, 141, 107685.\u003c/li\u003e\n\u003cli\u003eHe, L., Du, P., Chen, Y., Lu, H., Cheng, X., Chang, B., Wang, Z., 2017. Advances in microbial fuel cells for wastewater treatment. Renewable and Sustainable Energy Reviews, 71, 388-403.\u003c/li\u003e\n\u003cli\u003eHo, T.A., Criscenti, L.J., Wang, Y., 2016. Nanostructural control of methane release in kerogen and its implications to wellbore production decline. Sci Rep, 6, 28053.\u003c/li\u003e\n\u003cli\u003eHuang, T., Liu, W., Zhang, Y., Zhou, Q., Wu, Z., 2020. A stable simultaneous anammox, denitrifying anaerobic methane oxidation and denitrification process in integrated vertical constructed wetlands for slightly polluted wastewater. Environ. Pollut. 265, 119-128.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"7\"\u003e\n\u003cli\u003eKasak, K., Mander, \u0026Uuml;., Truu, J., Truu, M., J\u0026auml;rveoja, J., Maddison, M., Teemusk, A., 2015. Alternative filter material removes phosphorus and mitigates greenhouse gas emission in horizontal subsurface flow filters for wastewater treatment. Ecological Engineering, 77, 242-249.\u003c/li\u003e\n\u003cli\u003eKumar, S.S., Kumar, V., Gnaneswar Gude, V., Malyan, S.K., Pugazhendhi, A., 2020. Alkalinity and salinity favor bioelectricity generation potential of Clostridium, Tetrathiobacter and Desulfovibrio consortium in Microbial Fuel Cells (MFC) treating sulfate-laden wastewater. Bioresource Technology, 306, 123110.\u003c/li\u003e\n\u003cli\u003eLee, S.J., McCormick, M.S., Lippard, S.J., Cho, U., 2013. Control of substrate access to the active site in methane monooxygenase. Nature, 494, 380-384.\u003c/li\u003e\n\u003cli\u003eLi, H., Song, H., Yang, X., Zhang, S., Yang, Y., Zhang, L., Xu, H., Wang, Y., 2018. A continuous flow MFC-CW coupled with a biofilm electrode reactor to simultaneously attenuate sulfamethoxazole and its corresponding resistance genes. Science of the Total Environment, 637-638, 295-305.\u003c/li\u003e\n\u003cli\u003eLi, S., Hu, Z., Zhang, J., 2017. Management and high-value utilization of constructed wetland plants: an overview. Environmental pollution and prevention, 39, 432-438.\u003c/li\u003e\n\u003cli\u003eLi, W., Quan, X., Chen, L., Zheng, Y., 2020. Application of slow-release carbon sources embedded in polymer for stable and extended power generation in microbial fuel cells. Chemosphere, 244, 125515.\u003c/li\u003e\n\u003cli\u003eLiu, W., Xiao, H., Ma, H., Li, Y., Adyel, T.M., Zhai, J., 2020. Reduction of methane emissions from manganese-rich constructed wetlands: Role of manganese-dependent anaerobic methane oxidation. Chemical Engineering Journal, 387.\u003c/li\u003e\n\u003cli\u003eLu, L., Xing, D., Ren, Z.J., 2015. Microbial community structure accompanied with electricity production in a constructed wetland plant microbial fuel cell. Bioresource Technology, 195, 115-121.\u003c/li\u003e\n\u003cli\u003eMaltais-Landry, G., Maranger, R., Brisson, J., Chazarenc, F., 2009. Greenhouse gas production and efficiency of planted and artificially aerated constructed wetlands. Environmental Pollution, 157, 748-754.\u003c/li\u003e\n\u003cli\u003eMander, \u0026Uuml;., Dotro, G., Ebie, Y., Towprayoon, S., Chiemchaisri, C., Nogueira, S.F., Jamsranjav, B., Kasak, K., Truu, J., Tournebize, J., 2014. Greenhouse gas emission in constructed wetlands for wastewater treatment: A review. Ecological Engineering, 66, 19-35.\u003c/li\u003e\n\u003cli\u003eMander, \u0026Uuml;., Dotro, G., Ebie, Y., Towprayoon, S., Chiemchaisri, C., Nogueira, S.F., Jamsranjav, B., Kasak, K., Truu, J., Tournebize, J., Mitsch, W.J., 2014. Greenhouse gas emission in constructed wetlands for wastewater treatment: A review. Ecological Engineering, 66, 19-35.\u003c/li\u003e\n\u003cli\u003eMar\u0026iacute;a E, H., Michelli, G., Juan Carlos, H., 2017. Greenhouse gas emissions and pollutant removal in treatmentwetlands with ornamental plants under subtropical conditions. Ecological Engineering, 35, 164-152.\u003c/li\u003e\n\u003cli\u003eMeng, L., Li, X., Wang, S., Liu, L., Ma, K., Zhang, J., 2017. The long-term impact of cefalexin on organic substrate degradation and microbial community structure in EGSB system. Chemosphere, 184, 215223.\u003c/li\u003e\n\u003cli\u003eNie, W., Xie, G., Ding, J., Lu, Y., Liu, B., Xing, D., Wang, Q., Han, H., Yuan, Z., Ren, N., 2019. High performance nitrogen removal through integrating denitrifying anaerobic methane oxidation and Anammox: from enrichment to application. Environment International, 132, 105107.\u003c/li\u003e\n\u003cli\u003eOon, Y., Ong, S., Ho, L., Wong, Y., Dahalan, F.A., Oon, Y., Lehl, H.K., Thung, W., 2016. Synergistic effect of up-flow constructed wetland and microbial fuel cell for simultaneous wastewater treatment and energy recovery. Bioresource Technology, 203, 190-197.\u003c/li\u003e\n\u003cli\u003eRout, S., Parwaiz, S., Nayak, A.K., Varanasi, J.L., Pradhan, D., Das, D., 2020. Improved bioelectricity generation of air-cathode microbial fuel cell using sodium hexahydroxostannate as cathode catalyst. Journal of Power Sources, 450, 227679.\u003c/li\u003e\n\u003cli\u003eSemeraro, T., Giannuzzi, C., Beccarisi, L., Aretano, R., De Marco, A., Pasimeni, M.R., Zurlini, G., Petrosillo, I., 2015. A constructed treatment wetland as an opportunity to enhance biodiversity and ecosystem services. Ecological Engineering, 82, 517-526.\u003c/li\u003e\n\u003cli\u003eSong, Y., Wang, F., Liu, G., Luo, S., Guo, R., 2017. Controllable methane hydrate formation through trace carbon dioxide charging. Fuel, 203, 145-151.\u003c/li\u003e\n\u003cli\u003eSrivastava, P., Abbassi, R., Yadav, A.K., Garaniya, V., Asadnia, M., 2020. A review on the contribution of electron flow in electroactive wetlands: Electricity generation and enhanced wastewater treatment. Chemosphere, 254, 126926.\u003c/li\u003e\n\u003cli\u003eSrivastava, P., Yadav, A.K., Garaniya, V., Abbassi, R., 2019. Chapter 6.3 - Constructed Wetland Coupled Microbial Fuel Cell Technology: Development and Potential Applications. in: Mohan, S.V., Varjani, S., Pandey, A. (Eds.), Microbial Electrochemical Technology. Elsevier, pp. 1021-1036.\u003c/li\u003e\n\u003cli\u003eSu, J., Hu, C., Yan, X., Jin, Y., Chen, Z., Guan, Q., Wang, Y., Zhong, D., Jansson, C., Wang, F., Schn\u0026uuml;rer, A., Sun, C., 2015. Expression of barley SUSIBA2 transcription factor yields high-starch low-methane rice. Nature, 523, 602-606.\u003c/li\u003e\n\u003cli\u003eTang, C., Zhao, Y., Kang, C., Yang, Y., Morgan, D., Xu, L., 2019. Towards concurrent pollutants removal and high energy harvesting in a pilot-scale CW-MFC: Insight into the cathode conditions and electrodes connection. Chemical Engineering Journal, 373, 150-160.\u003c/li\u003e\n\u003cli\u003eVymazal, J., 2011. Constructed Wetlands for Wastewater Treatment: Five Decades of Experience. Environmental Science \u0026amp; Technology, 45, 61-69.\u003c/li\u003e\n\u003cli\u003eWang, J., Song, X., Wang, Y., Abayneh, B., Ding, Y., Yan, D., Bai, J., 2016. Microbial community structure of different electrode materials in constructed wetland incorporating microbial fuel cell. Bioresource Technology, 221, 697-702.\u003c/li\u003e\n\u003cli\u003eWang, J., Song, X., Wang, Y., Abayneh, B., Li, Y., Yan, D., Bai, J., 2016. Nitrate removal and bioenergy production in constructed wetland coupled with microbial fuel cell: Establishment of electrochemically active bacteria community on anode. Bioresource Technology, 221, 358-365.\u003c/li\u003e\n\u003cli\u003eWang, J., Song, X., Wang, Y., Bai, J., Li, M., Dong, G., Lin, F., Lv, Y., Yan, D., 2017. Bioenergy generation and rhizodegradation as affected by microbial community distribution in a coupled constructed wetland-microbial fuel cell system associated with three macrophytes. Science of the Total Environment, 607-608, 53-62.\u003c/li\u003e\n\u003cli\u003eWang, J., Song, X., Wang, Y., Zhao, Z., Wang, B., Yan, D., 2017. Effects of electrode material and substrate concentration on the bioenergy output and wastewater treatment in air-cathode microbial fuel cell integrating with constructed wetland. Ecological Engineering, 99, 191-198.\u003c/li\u003e\n\u003cli\u003eWang, X., Daigger, G., Lee, D., Liu, J., Ren, N., Qu, J., Liu, G., Butler, D., 2018. Evolving wastewater infrastructure paradigm to enhance harmony with nature. Science Advances, 4.\u003c/li\u003e\n\u003cli\u003eWeaver, A.J., 2011. Toward the Second Commitment Period of the Kyoto Protocol. Science 332 (6031), 795-796.\u003c/li\u003e\n\u003cli\u003eWetser, K., Sudirjo, E., Buisman, C.J.N., Strik, D.P.B.T., 2015. Electricity generation by a plant microbial fuel cell with an integrated oxygen reducing biocathode. Applied Energy, 137, 151-157.\u003c/li\u003e\n\u003cli\u003eXiao, N., Wu, R., Huang, J.J., Selvaganapathy, P.R., 2020. Anode surface modification regulates biofilm community population and the performance of micro-MFC based biochemical oxygen demand sensor. Chemical Engineering Science, 221, 115691.\u003c/li\u003e\n\u003cli\u003eXu, L., Zhao, Y., Tang, C., Doherty, L., 2018. Influence of glass wool as separator on bioelectricity generation in a constructed wetland-microbial fuel cell. Journal of Environmental Management, 207, 116-123.\u003c/li\u003e\n\u003cli\u003eYadav, A.K., Dash, P., Mohanty, A., Abbassi, R., Mishra, B.K., 2012. Performance assessment of innovative constructed wetland-microbial fuel cell for electricity production and dye removal. Ecological Engineering, 47, 126-131.\u003c/li\u003e\n\u003cli\u003eYang, H., Bao, B., Liu, J., Qin, Y., Wang, Y., Su, K., Han, J., Mu, Y., 2018. Temperature dependence of bioelectrochemical CO2 conversion and methane production with a mixed-culture biocathode. Bioelectrochemistry, 119, 180-188.\u003c/li\u003e\n\u003cli\u003eYousefi, V., Mohebbi-Kalhori, D., Samimi, A., 2017. Ceramic-based microbial fuel cells (MFCs): A review. International Journal of Hydrogen Energy, 42, 1672-1690.\u003c/li\u003e\n\u003cli\u003eZhang, K., Liu, Y., Chen, Q., Luo, H., Zhu, Z., Chen, W., Chen, J., Mo, Y., 2018. Effect of submerged plant species on CH4 flux and methanogenic community dynamics in a full-scale constructed wetland. Ecological Engineering, 115, 96-104.\u003c/li\u003e\n\u003cli\u003eZhang, K., Wu, X., Luo, H., Li, X., Chen, W., Chen, J., Mo, Y., Wang, W., 2020. CH4 control and associated microbial process from constructed wetland (CW) by microbial fuel cells (MFC). Journal of Environmental Management, 260, 110071.\u003c/li\u003e\n\u003cli\u003eZhang, S., You, J., Chen, H., Ye, J., Cheng, Z., Chen, J., 2020. Gaseous toluene, ethylbenzene, and xylene mixture removal in a microbial fuel cell: Performance, biofilm characteristics, and mechanisms. Chemical Engineering Journal, 386, 123916.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CW-MFC, CH4 control, Bioelectricity generation, Plant root location, Biochemical mechanism","lastPublishedDoi":"10.21203/rs.3.rs-202429/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-202429/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the context of global warming, the bioelectrochemical method (microbial fuel cell MFC) was proposed for CH\u003csub\u003e4\u003c/sub\u003e control from CWs. The main focus is to further explore the effect of plant roots location at the electrode, plant species on CH\u003csub\u003e4\u003c/sub\u003e emissions, bioelectricity generation and the mechanism underlying competition between electrogenesis and methanogenesis at the anode. The results showed that the operation of MFC effectively reduced the CH\u003csub\u003e4\u003c/sub\u003e emissions and promoted COD removal rates. CH\u003csub\u003e4\u003c/sub\u003e emission was significantly higher in open circuit (6.2 mg m\u003csup\u003e-2\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) than in closed circuit reactors (3.1 mg m\u003csup\u003e-2\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e). Plant roots at the cathode had the highest electricity generation and the lowest CH\u003csub\u003e4\u003c/sub\u003e emissions. The highest power generation (0.49 V, 0.33 w m\u003csup\u003e-3\u003c/sup\u003e) and the lowest CH\u003csub\u003e4\u003c/sub\u003e emissions (2.3 mg m\u003csup\u003e-2\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) were observed in the reactors where \u003cem\u003eTypha Orientalis \u003c/em\u003ewas planted with plant roots at the cathode. The role of plants in strengthening electron acceptor was greater than that of plant rhizodeposits in strengthening electron donors. q-PCR and correlation analysis indicated that the \u003cem\u003emcr\u003c/em\u003eA genes and CH\u003csub\u003e4\u003c/sub\u003e emissions were positively correlated (\u003cem\u003er=0.98, p\u0026lt;0.01\u003c/em\u003e), while no significant relationship between CH\u003csub\u003e4\u003c/sub\u003e emissions and \u003cem\u003epmoA \u003c/em\u003egenes was observed. More nanowires, which are conductive to electron transfer, were found when plant roots were in cathode by scanning electron microscope (SEM). Illumina sequencing revealed that more abundant exoelectrogens and denitrifying bacteria (\u003cem\u003eGeobacter\u003c/em\u003e, \u003cem\u003eDesulfobulbu, Nitrospira \u003c/em\u003eand\u003cem\u003e Anaerolinea)\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003ewere observed when plant roots located in cathodes. Strictly acetotrophic archae (Methanosaetaceae) were likely main electron donor competitors with exoelectrogens. In addition, plant species played a more important role in CH\u003csub\u003e4\u003c/sub\u003e emissions and electricity generation than the plant roots location at the electrode. Therefore, it is necessary to strengthen plant configuration to reduce CH\u003csub\u003e4\u003c/sub\u003e emissions, so as to promote sustainable development of wastewater treatment. \u003c/p\u003e","manuscriptTitle":"Application of Bioelectrochemical Technique in Energy Recovery and CH4 Control From Constructed Wetland (CW) and Associated Biochemical Mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-17 22:46:34","doi":"10.21203/rs.3.rs-202429/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92cd895c-b0b1-4dcc-b1a8-71616d696ca9","owner":[],"postedDate":"February 17th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2466298,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-05-24T11:42:00+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-17 22:46:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-202429","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-202429","identity":"rs-202429","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}