3.1. Methane fluxes in closed/open circuit mode CW-MFCs
The open/closed circuit model is one of the most important factors affecting CH4 emissions. In the opened circuit case, the CW-MFC is equivalent to a CW, so it is important to explore the relationship between the two, the dynamics of the two modes of open/closed circuit during operation are shown in Fig. 2(A). Compared the analysis of with plants, the CH4 emission flux in the opened circuit mode was higher than closed circuit mode by about 0.46 ± 0.024 mg/(m2·h). In the system non-plants, the CH4 emission flux in the opened circuit mode was higher than closed circuit mode by about 0.21 ± 0.012 mg/(m2·h), which clearly shown that the CH4 emission from the opened circuit is higher than closed circuit. In previous studies, the CH4 emission fluxes were 6.37–7.28 mg/(m2·h) and 7.43–8.36 mg/(m2·h) for closed circuit and opened circuit, respectively, and the CH4 emission fluxes in our study was basically consistent with the relevant literature report(Xu, Song et al. 2021). The difference between the open/closed circuit model is the bioanode. It has been reported in the literature that electrons are more easily produced in closed circuit mode due to electrical stimulation to produce electrochemically active bacteria (EAB), with the same source of carbon and nitrogen, electrogenic bacteria have easier access to food, allowing methanogenic bacteria to be suppressed(Kaur, Boghani et al. 2014). In an anaerobic fermentation environment, no current passes in the open circuit, methanogenic bacteria proliferate and increase the production of CH4 gas(Ishii, Hotta et al. 2008).
Of course, the effect of substrate concentration on GHG emissions were exceptionally important, the higher the substrate concentration, the more microorganisms would be on the anode(Rahmani, Navidjouy et al. 2022). However, considered the effect of plants on CH4 gas emissions, i.e., controlled the glucose concentration at 200 mg/L, it was found that plants also increased CH4 gas emissions. In the closed circuit system, the plant group improved by about 0.48 ± 0.021 mg/(m2·h), compared to the non-plant group, the plant roots (cellulose) were easily decomposed by microorganisms for their own consumption, increasing the CH4 emission(Rismani-Yazdi, Carver et al. 2013). Moreover, when plants were planted, GHG emissions were not only in the form of gas spillage and liquid-phase diffusion, but also in the form of gas transport through plant pipes(Gong, Song et al. 2020). Excluding the effect of substrate concentration (i.e., entering substrate concentration of 0 mg/L) as shown specifically in Fig. 3, the CH4 emission flux of 0.39 ± 0.01 mg/(m2·h) was found for the plant group under closed circuit conditions. In contrast, the microorganisms on the anode of the plant-free group in the absence of substrate concentration had no nutrients and died. The fact that cellulose can be used as a carbon source by cellulomonas fimi, cellulomonas biazotea, and cellulomonas flavigena, where cellulomonas spp. is a direct cellulose-based microorganism(Takeuchi, Khawdas et al. 2017, Toczylowska-Maminska, Szymona et al. 2018).
In addition, the relationship between CH4 gas emissions and output voltage can be further illustrated with polarization curves and power density curves. As shown in Fig. 2(B), the current density of the plant group was 1.07 W/m3 higher than the plant-free group, corresponding to an internal resistance of 187.02 Ω and257.91.9 Ω, respectively. This also indicated that the addition of plant roots provides more terminal reduction electron acceptors (O2) and increases the reduction medium on the cathode surface reducing the internal resistance of the system thus increasing the output voltage, however, the plant causes an increase in the CH4 emission flux(Nandy, Sharma et al. 2019). From a microbiological point of view, electrochemical bacteria (e.g., Aspergillus, Actinobacter, Fimicus and Acidobacter) on the anode compete with methanogenic bacteria for nutrients, electrogenic bacteria become the dominant flora resulting in increased voltage and thus inhibiting CH4 production. The same explains the transport of CH4 gas in vascular tissues inside the plant, which increases the emission of the gas. Rice as a typical vascular plant, according to the literature, the power density with and without plants was 26 ± 7 mW/m2 and 3.7 ± 1.8 mW/m2, respectively, while rice wetlands induced CO2 as well as CH4 production(De Schamphelaire, Van den Bossche et al. 2008, Wang, Yu et al. 2018). The current density in the plant group without substrate can reach 1.43 A/m3 and the power density is 0.5 W/m3, yet the internal resistance is as high as 233.15 Ω. The reason for this phenomenon may be a decrease in substrate concentration, microorganisms do not have enough nutrients for their own needs thus reducing the production of electrons and making it difficult for protons to pass from the anode to the cathode. It has been reported in the literature that cellulose can only recharge the microorganisms in MFCs, especially the Cellulomonas strain NBRC-15513(Khawdas, Watanabe et al. 2019).
3.2. CH4 emission fluxes in different external resistances
The external resistance can affect the stable output of voltage and the best treatment of wastewater. Therefore, different external resistances (100 Ω, 200 Ω, 500 Ω and 1000 Ω) are set according to the purpose of the experiment to observe the control of GHG. As shown in Fig. 3(A), with the external resistance increases, the emission trend of CH4 also increases gradually. Compared external resistance 100 Ω and 1000 Ω respectively, the 1000 Ω increased by about 0.669 + 0.012 mg/(m2·h). It was enough to show that the external resistance was in positive proportion to CH4 emission, but it has no statistical significance in practice. It was further understood from the figure that different external resistance has different control over methane, because the electron transfer rate, changes of microbial metabolic activities and kinetics of substrate utilization are different under different external resistance(Picioreanu, Head et al. 2007). Therefore, it was further considered to increase the external resistance and increase the growth of methanogens, which is not the growth of electrogenic bacteria, but the growth of electroactive microorganisms mainly uses the substrate under the action of electrical stimulation(Picioreanu, van Loosdrecht et al. 2008).
As shown in Fig. 3(B), when the external resistance was 1000 Ω, the power density reaches the maximum (the maximum power density was 2.17 A/m3), but the current gradually decreases, making CH4 reach the maximum. However, when the external resistance was 100 Ω, the power density reaches 1.38 A/m3, the current is increasing, and methane was well controlled. This was enough to show that there is a strong competitive relationship between the electrogenic bacteria on the anode and the methanogenic bacteria to produce current by consuming the substrate. Of course, the increase of external resistance reduces the degradation of COD removal rate from 95.71–90.12%. This may be because the high external resistance is not conducive to the consumption of organic matter by anode microorganisms, reduces the ability of microorganisms to produce electrons, and increases the emission flux of CH4.
3.3. The ultimate fate of "carbon cycle"
The question of the final destination of the wetland "carbon cycle" is to explore the key process of carbon conversion in wetlands, which has important implications for the carbon saving function. The experiments in this section describe the transport and transformation of wetland carbon fractions between different interfaces in the atmospheric vegetation, water bodies and summarize the carbon cycle in terms of the carbon stocks accounted for by the atmosphere, water bodies and plants.
TC (glucose) enters as an aqueous solution (TC ~ 606.9 mg/L), it is transported and transformed under anaerobic and microbial conditions or is also broken down and transformed by cellulosic bacteria in the plant root system (element [C] is in the form of small organic molecules), the above process has both liquid-liquid transfer and gas-liquid exchange([C] is in the form of gas and organic matter). Of course, the aeration tissue of plant roots also transmits a small amount of gas to the atmosphere, and the total emission fluxes of CO2 and CH4 are 62.76 mg and 25.84 mg, and the final TC of the effluent water was 352.19 mg/L. As shown in Fig. 4, a large amount of substrate is consumed by microorganisms in solution, accounting for about 40.28% of the entire system, CH4 and CO2 account for 11.16% and 24.6% of the entire graph, respectively, indicating a larger production of gases, the remainder is influenced by other factors. The carbon content in the matrix was complex, accounting for about 10.76% of the pie chart, including microorganisms in the matrix and cellulose shed by plant roots. The carbon content of plants also changed before and after the reaction, accounting for about9.34%.
The substrate concentration only provides nutrients to the microorganisms, and the microorganisms' extracellular electron transfer capacity is enhanced, thus favoring the development of electrogenic bacteria and thus inhibiting methane production, so the substrate concentration occupies a large part. Due to the temporal and spatial variability of greenhouse gas emissions from wetlands, the flux of CH4 emissions increases as the flux of CO2 emissions increases, because the increase in CO2 concentration changes the concentration of oxygen in the root zone and the availability of carbon sources(Kao-Kniffin and Zhu 2013, Zhang, Brodylo et al. 2021). The addition of plants increases the gas transmission and small molecules of organic matter (a few small molecules) are absorbed by the plants to purify the wastewater and increase the voltage of the system. Using carbon (C) and nitrogen (N) isotopic signatures of organic matter (OM) to detect changes in associated plant and microbial processes in aquatic systems(Olaleye, Nkheloane et al. 2014). In contrast, carbon isotopes are variable in wetland plants, including photosynthetic pathways, the nature of the major inorganic carbon sources (atmospheric and dissolved), the form of effective carbon and subsequent assimilation (CO2, HCO3−), and the basis for diffusion limitations by plant life forms or aquatic environmental conditions(Guareschi, Pereira et al. 2014). In summary, it can be seen that the tendency of [C] elements can be roughly divided into those consumed by microorganisms, those produced by liquid-gas conversion and gas-gas exchange, and finally, the nutrient solution coming out of the cathode port before the substrate is consumed.
3.4. Bacterial community analysis
3.4.1 Richness and diversity of the microbial community
The data of bacterial community of wetland microbial dye battery to control GHGs emission are limited, so it is of great significance to analyze the richness and diversity of bacterial community(Lopez, Sepulveda-Mardones et al. 2019). As shown in Table 1, Chao1, Shannon, observed species and Simpson shown that the total number of bacteria and species richness of CCP were higher than those of CCN and of OCP. This indicated that the closed circuit helps the electron-producing bacteria to produce electrons and the plants planted at the anode help to enrich the bacterial community leading to an increase in both electricity and gas production. And Faith-pd and Pielou-e in the table illustrate that the higher their values, the better the genetic diversity of the species and the homogeneity of the community. The above phenomenon is reflected in 3.1 and 3.2 where the addition of plants contributes to the enhancement of the bacterial community. Of course, plant roots help microorganisms to adhere and obtain nutrients so that bacteria accelerate extracellular electron transfer(Zhang, Liang et al. 2016).
3.4.2 Methanogenic bacteria community
Further investigation of the effect of open and closed circuit and the presence or absence of plants on the bacterial community composition at the anode gate level of the system is shown in Fig. 5. In the plant closed-circuit system, the dominant bacteria are Acinetobacter, Pseudomonas, spirochetes and Bacteroides, accounting for 1.85%, 18.77%, 7.54% and 7.79% respectively. In the opened circuit system with plants, the dominant bacteria are Acinetobacter and spirochetes, accounting for 78.77% and 3.29% respectively. In the non-plant closed circuit, the dominant bacteria were Acinetobacter, Pseudomonas, spirochetes and Bacteroides, accounting for 1.25%, 6.16%, 7.28% and 7.68% respectively. Through comparative analysis, under the conditions of open and closed circuit in plants, it can be found that Acinetobacter is suitable to survive in the case of open circuit, and the electricity producing microorganism is relatively single. The richness of microbial flora in closed circuit is higher than that in open circuit, which further proves that the diversity of electrogenic bacteria in closed plant system in 3.4.1 is richer, and only Acinetobacter was in open circuit. Compared with the situation with or without plants in the closed system, the richness of microbial flora is richer than that without plants, which may be because the addition of plants increases the diversity of flora. From the perspective of relative richness, the richness of the plant group is slightly higher than that of the non-plant group, which also greatly shows that the plant root system improves the nutrients for microorganisms, stimulates its reproduction rate, and makes the power production of the plant group higher than that of the non-plant group.
Methanogenic bacteria are the main genus of methane producing bacteria, as shown in Fig. 6, the main bacteria producing methane were Methanothrix, Methanobacterium and Methanolinea. All of the above bacteria are the main microorganisms producing methane. Comparative analysis shown that under the conditions of plant closed circuit and open circuit, the number of Methanothrix, Methanobacterium and Methanolinea in closed circuit is about 1%, 2.79% and 0.36% higher than that in open circuit, which fully shown that the number of methanogens in open circuit is higher, which is helpful for methanogens to produce CH4. By comparing the presence or absence of plants in the closed circuit, it can be known that the methane emission flux of Methanothrix in the case of plants is about 4.74% higher than that in the non-plant group, which also greatly proves that the CH4 emission flux of closed circuit non plant in Section 3.1 was the smallest. However, Methanobacterium was higher in the closed-circuit plant-free group, with a value of about 2.44%, which may be because Methanogens was suitable to take the easily decomposed carbon source as the nutrient, while cellulose is a macromolecular organic matter, which is difficult to decompose. The coexistence of Methanothrix and MethanoRegula can be concluded from the analysis, which indicated that methane gas production greatly originates from wetlands, and the use of acetate and CO2/H2 were two common substrates(Galand, Fritze et al. 2005). The above detection of methanogens at the genus level in 16S does not prove the relative richness of electrogenic bacteria and methanogens at the genus level, which needs to be further explored.
3.5. Mechanism of methane emission
To investigate the mechanism of electricity production by bioanodes, the competition mechanism between electricity-producing bacteria and methanogenic bacteria was further explored from the above study, as well as the analysis of the principle of methane gas production. CH4 is not produced under all conditions, but rather due to the limited oxygen supply and anaerobic state of the wetland, which creates the prerequisites for wetland methane production(de la Varga, Ruiz et al. 2015). Of course, CH4 production can be divided into three stages, the first is the anaerobic fermentation and decomposition of complex organic substrates by fermenting bacteria into ethanol and fatty acids, etc., and the production of acetic acid, hydrogen and CO2 by syntrophic bacteria. Or acetic acid production by specialized acetic acid-producing bacteria, and finally methane production from acetic acid or CO2/H2 in the presence of methanogenic bacteria(Wang and Ren 2013). The specific equation is as follows.
C6H12O6 + 2H2O → 2CO2 + 2CH3COOH + 4H2 (1)
CH3COOH → CO2 + CH4 (2)
4HCOOH → CH4 + 3CO2 + 2H2O (3)
4CH3OH → 3CH4 + CO2 + 2H2O (4)
4CO + 2H2O → CH4 + 3CO2 (5)
CO2 + 4H2 → CH4 + 2H2O (6)
The contribution of the two CH4 production methods varies due to differences in microbial families, organic matter species and content, etc. in different wetlands. A study of peat bogs using isotope tracing found that 70% of the CH4 was produced by fermentation of acetic acid, while only 30% was formed by reduction of CO2(Chen, Zhao et al. 2021). In contrast, the root system in the plant group, the plant has an enhanced ability to deliver oxygen to the cathode, resulting in an enhanced ability to produce H2O from the cathode. However, the oxygen-secreting capacity of the root system shows a positive correlation with the aeration tissue, so not only does the concentration of the carbon source increase in the presence of plants, but their aeration tissue also emits a certain amount of methane gas.