Performance and mechanism for efficient dechlorination in 3D-macroporous-bioanode-installed microbial fuel cells

Performance and mechanism for efficient dechlorination in 3D-macroporous-bioanode-installed microbial fuel cells | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 September 2025 V1 Latest version Share on Performance and mechanism for efficient dechlorination in 3D-macroporous-bioanode-installed microbial fuel cells Author : JuP YOU 0000-0003-2714-5368 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175727534.47607188/v1 160 views 116 downloads Contents Abstract Abstract 1. Introduction 2. Results and Discussion 3. Conclusion 4. Experimental Section Refernces Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Anode morphology is the main factor influencing the removal of chlorine-containing pollutants through promising microbial fuel cells (MFCs). Large-pore three-dimensional sponge-like anodes (nickel foam [NF], carbon felt [CF], and reticulated vitreous carbon [RVC]), are used as metal-, carbon-, and carbon–metal-based materials. Compared to MFC-NF and MFC-CF, the output voltage (55% and 61%, respectively) and average chlorobenzene (CB) degradation rate (285.4% and 52.8%, respectively) of MFC-RVC are higher. Meanwhile, the coulombic efficiency reaches a maximum (12.4%), while the dechlorination efficiency reaches 95.6%. The outperformance of MFC-RVC is related to its higher resistivity (5.8 × 10−6 Ω m; 1.6-fold that of CF, and close to that of NF) as well as its specific surface area and hydrophilic properties. The amounts of biomass and CB degraders (Actinobacteria) in the RVC biofilm greatly exceeded those of the other two anodes, with living microorganisms maintained at almost 88% after long-term operation, and the cellular activity reaching a maximum (10.80 U g−1 and 105.13 nmol L−1 for dehydrogenase and cytochrome C, respectively). Additionally, the mechanism underlying enhanced CB degradation by carbon–metal RVC anodes is elucidated by comparing Cl- and CO2 production, and suggestions for electrode selection, alongside guidance for CB removal in bioelectrochemical systems, are provided. Article category: Regular Article Subcategory: microbial fuel cells Performance and mechanism for efficient dechlorination in 3D-macroporous-bioanode-installed microbial fuel cells jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Zihan Song, Lvzheng Lai, Zanyun Ying, Jingkai Zhao, Dongzi Chen*, Juping You* Z. Song, L. Lai, D. Chen, J. You Zhejiang Key Laboratory of Pollution Control for Port-Petrochemical Industry, Zhejiang Ocean University, Zhoushan 316022, China E-mail: [email protected] Z. Ying Ningbo Key Laboratory of Agricultural Germplasm Resources Mining and Environmental Regulation, College of Science & Technology, Ningbo University, Ningbo 315212, China J. Zhao College of Environment, Zhejiang University of Technology, Hangzhou 310014, China Keywords: chlorobenzene removal, microporous anodes, microbial fuel cells, mechanisms jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Abstract Anode morphology is the main factor influencing the removal of chlorine-containing pollutants through promising microbial fuel cells (MFCs). Large-pore three-dimensional sponge-like anodes (nickel foam [NF], carbon felt [CF], and reticulated vitreous carbon [RVC]), are used as metal-, carbon-, and carbon–metal-based materials. Compared to MFC-NF and MFC-CF, the output voltage (55% and 61%, respectively) and average chlorobenzene (CB) degradation rate (285.4% and 52.8%, respectively) of MFC-RVC are higher. Meanwhile, the coulombic efficiency reaches a maximum (12.4%), while the dechlorination efficiency reaches 95.6%. The outperformance of MFC-RVC is related to its higher resistivity (5.8 × 10 −6 Ω m; 1.6-fold that of CF, and close to that of NF) as well as its specific surface area and hydrophilic properties. The amounts of biomass and CB degraders (Actinobacteria) in the RVC biofilm greatly exceeded those of the other two anodes, with living microorganisms maintained at almost 88% after long-term operation, and the cellular activity reaching a maximum (10.80 U g −1 and 105.13 nmol L −1 for dehydrogenase and cytochrome C, respectively). Additionally, the mechanism underlying enhanced CB degradation by carbon–metal RVC anodes is elucidated by comparing Cl - and CO 2 production, and suggestions for electrode selection, alongside guidance for CB removal in bioelectrochemical systems, are provided. 1. Introduction Chlorine-containing volatile organic compounds (Cl-VOCs) are typical atmospheric pollutants emitted by chemical and pharmaceutical industries. [1] Most Cl-VOCs, including chlorobenzene (CB), dichloromethane, and chloroethane, are not only recognized as major contributors to the formation of ozone, secondary aerosols, and photochemical smog, [2] In the recently promulgated Chain list of toxic and harmful air pollutants, Cl-VOCs account for 4 out of 11, and Cl-VOCs have attracted increased attention from environmental researchers. Therefore, given the stringent national and local emission standards, the effective elimination of Cl-VOCs is imperative and urgently required. Technologies such as thermal oxidation, adsorption, low-temperature plasma treatment, and photocatalysis have been widely used for Cl-VOC abatement. [3-9] Although these methods are effective for Cl-VOC control, they are associated with issues such as high risks, high costs, insufficient efficiency, and secondary pollution, especially the production of dioxins during combustion. [10] Biological methods, with the advantages of eco-friendliness, low costs, and complete degradation, have been developing rapidly. [11] However, the presence of Cl atom rings passivates substitution reactions, making breakage of C-Cl bonds difficult for microorganisms in the natural environment, and the biological toxicity of Cl-VOCs has a negative impact on biotreatment processes. [12] The high Henry constant of Cl-VOCs is an additional factor that limits their biodegradation. [13] Wu et al. evaluated the biodegradation of 1,2-dichloroethane in airlift bioreactors and found that the removal efficiency in an airlift bioreactor with 7% silicone oil was only 46.9 ± 2.4% at an empty-bed residence time of 75 s and inlet loading rate of 10.7 ± 2 g m −3 h −1 . This indicated that the activity of microorganisms may play a more important role in the degradation of Cl-VOCs. [14] A bioelectrochemical system (BES), based on positively enhances an electrode and microorganisms, positively enhances the oxidation reaction at the anode or the reduction reaction at the cathode. [15,16,17] Over the years, the removal of Cl-VOCs by the biocathodes of microbial electrolytic cells (MECs) has been investigated extensively. [18,19] Ying et al. [20] indicated that the removal efficiency of 300~1200-mg m −3 CB in MECs exceeded 75%, the maximum CB removal capacity was 322.58 mg m −3 s −1 , and both the removal efficiency and elimination capacity were superior to those of biofilter and UV-biofilter systems. The authors also added anthraquinone-2,6-disulfonate (AQDS) and flavin mononucleotide (FMN) as redox mediators to enhance electron transfer between the biofilm and electrode and found that the removal efficiency of CB in MEC-AQDS and MEC-FMN increased by 40% and 20%, respectively. In particular, the dechlorination and Coulombic efficiencies of MEC-AQDS were 66% and 50% higher than those of MEC-FMN, respectively. [21] Microbial fuel cells (MFCs), the most renowned type of BES, not only accelerate biodegradation but also directly convert chemical energy stored in organics (Cl-VOCs) to electricity. [22,23] Moreover, they are considered a more energy-efficient technology compared to MEC. 1,2-dichloroethane has been efficiently degraded in MFCs at a rate of 102 mg L −1 d −1 , and the energy released from this degradation was partially recovered as electricity (43%). [24] Notably, the activity of electroactive biofilm forms the cornerstone of MFC electro-microbial processes. [25] However, the spatial distribution and activity of biofilm is significantly influenced by the pore sizes of anodes, a few micrometers (below 10 μm) could enable microbial cells to penetrate but do not favour for biofilm formation, a few tens of micrometers (10 to 99 μm) are subject to clogging, and a few hundreds of pore sizes allows cells adhesion inside of the anode structure, but its development may limited by internal mass transport and local acidification inside the porous structure. [26] Thus, anode characteristics are often explored in terms of contaminant biodegradation and current density blossoming. Consequently, studies are increasingly focusing on three-dimensional (3D) macroporous electrodes. The skeleton of a 3D anode was interspersed with microbial layers to create a ”sponge-like” biofilm, which provided a larger specific surface for loading active microbial species and making up for the drawbacks of mesoporous electrodes. [27,28] Carbonized loofah sponges, carbonized mushrooms, carbonized pomelos, carbonized textiles, reticulated vitreous carbon (RVC) foam, Ni foam (NF), stainless-steel foam graphene sponges, carbon nanotube sponges, and carbon sponges, among other materials, with pore sizes ranging from 100 to 1000 μm are suitable macroporous anodes for forming 3D biofilm. [26,29-32] Metal-based materials exhibit high electrical conductivity and superior mechanical properties, and they have been used as anodes in various MFCs. [33] Some precious metals, such as gold and silver, have demonstrated excellent electrical energy conversion capabilities. [34] However, the high cost of precious metals hinders their practical application. Other metal anodes, such as copper, stainless steel, titanium, aluminum, and nickel, also have drawbacks in terms of microbial growth and colonization, particularly the exudation of metal ions during long-term operation. [35] The conductivity of most carbon materials are lower than that of metal substrates; however, recent studies have shown that carbon-based anodes provide a conductive microenvironment for biofilm formation. [36,37,38] To reduce the corrosiveness of metals while maintaining the high bioaffinity of carbon materials, metal electrodes can be modified with carbon materials; another method is to modify carbon materials with metal ions. [39,40,41] Studies on the influence of 3D macroporous carbon and metal based anodes on Cl-VOC treatment in MFCs are limited, and the differences among 3D metal, carbon, and carbon-metal anodes in terms of dechlorination, along with the underlying mechanisms, require further investigation. Therefore, considering material properties, conductivity, cost, bioaffinity, and hydrophilicity, three large commercial 3D macroporous materials, namely NF, carbon felt (CF), and RVC, were selected as representative metal, carbon, and carbon–metal anodes to enhance the dechlorination of Cl-VOCs. The surface properties and electrochemical characteristics of the anodes were analyzed, and the removal efficiency of a representative CB, as well as the energy recovery of MFCs, were investigated and compared. Moreover, the spatial distribution of live bacteria, the composition of the microbial community, and its metabolic pathways were explored to clarify the dechlorination mechanism of 3D anode-based MFCs in VOC biodegradation. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf 2. Results and Discussion 2.1. Properties of Anode Materials The 3D morphology and porous structure, revealing a large pore size, were monitored for all three anodes, after which Scanning Electron Microscope images of the three naked anodes were amplified and compared. The skeletons of the RVC and NF were found to be more robust and coarser than that of the fibrous CF, indicating that better conditions were provided for microbial attachment ( Figures 1 a–c). The resistivities of the NF, CF and RVC anodes were 6.7 × 10 −6 Ω m, 3.8 × 10 −3 Ω m and 5.8 × 10 −6 Ω m, respectively, which suggested that the electrical conductivities of NF and RVC exceeded that of CF. [42] Furthermore, the contact angles of the NF, CF, and RVC were 86.1°, 108.3°, and 71.9°, respectively (Figures 1d–f), indicating that the hydrophilicity of the three anodes followed the order of RVC > NF > CF, with high hydrophilicity favoring microbial attachment and biofilm formation. [43] The crystal structures are shown in Figure 1 g. In both the NF and RVC anodes, the three main diffraction peaks, located at 44°, 52°, and 76°, were attributed to the (111), (200), and (220) peaks, respectively, which is consistent with Ni, as evidenced by JCPDS 04-0850. [44] Amorphous carbon peaks, located at 26.8°, were detected in the CF and RVC, [45] and no other peaks were observed, which indicated that the purity of the selected NF and CF was acceptable. The values of the C and Ni peaks in the RVC pattern were lower than those in the CF and NF, because of the fact that only 8−10% of nickel was incorporated when preparing the RVC. This caused higher biocompatibility compared to that of NF and superior electrical conductivity compared with that of CF. [46] Information regarding the pore size of the three anodes is provided in Figures 1h and 1i. The smallest pore diameter among the three materials was 50 nm, and more than 90% of the pore diameters were above 100 nm, indicating that the selected NF, CF, and RVC were all macroporous materials. [47] The average pore diameters of the NF, CF and RVC were 550~650 μm, 50~120 μm, and 240~270 μm respectively. In addition, studies have shown that large pore sizes (of several hundred microns) allow microorganisms to enter 3D anodes, and further facilitate the development of active biofilms. 2.2. Start-Up and CB Removal in MFCs During the initial start-up stage, additional sodium lactate was provided to facilitate biofilm formation, after which CB alone served as a carbon source. As shown in Figure 2 a, when the removal efficiency of 50-mg L −1 CB exceeded 90% and the output voltages of MFC-NF, MFC-CF, and MFC-RVC were continuous and constant (at 106.5 mV, 160.7 mV, and 100.2 mV, respectively) over two consecutive cycles, the MFCs were successfully constructed and operated. The MFC-RVC was start-up successfully within shortest time (on day 35.0), followed by the MFC-CF (after 36.3 d) and then the MFC-NF (within 38.5 d). Because of the higher bio-impedance of CB, the start-up was slower than that of the MFCs driven by readily biodegraded toluene and ethyl acetate. [48,49] At CB concentrations of 50, 100, 150, and 200 mg L −1 , the output voltages reached maximums of 108.8 mV, 140.7 mV, 154.2 mV, and 77.6 mV for MFC-NF; 157.8 mV, 175.2 mV, 160.7 mV, and 102.1 mV for MFC-CF; and 108.3 mV, 248.3 mV, 182.5 mV, and 104.9 mV for MFC-RVC, respectively. The output voltages of the three MFCs increased as the CB concentration increased from 50 to 150 mg L −1 , and then decreased to the lowest value at the 200-mg L −1 feed stage. Notably, the MFC-RVC exhibited the highest value and the longest “plateau” on the output voltage curve, compared to MFC-NF and MFC-CF, indicating a higher electron recycling efficiency, [50] which was made possible by the strong conductivity of the RVC anode. The CB degradation performances of the three MFCs are shown in Figure 2 b. Under various CB conditions, the highest average CB degradation rates were achieved in the RVC-installed MFC. At CB concentrations of 50, 100, 150, and 200 mg L −1 , the average CB degradation rate of the MFC-RVC was 42.4%, 84.1%, 86.7%, and 285.4% higher than that of the MFC-NF and 0.4%, 7.2%, 21.0%, and 52.8% higher than that of the MFC-CF, respectively. In all three MFCs, the degradation capacity was the highest at 150-mg L −1 CB, with values of 14.4 mg L −1 h −1 , 11.9 mg L −1 h −1 , and 7.7 mg L −1 h −1 , respectively. A higher amount of CB (200 mg L −1 ) weakened the microbial activity, owing to its biological toxicity, and resulted in the lowest CB removal efficiency. [51] However, the removal capacity obtained in this study was superior to that of biotrickling filters (8.26 mg L −1 h −1 ) and MEC systems (12.9 mg L −1 h −1 ). [52,53] Thus, compared to traditional biological technology, MFC-RVC shows promising prospects for the degradation of CB while achieving electrical energy recovery. 2.3. Electrochemical Properties Power density and polarization curves for the MFCs (equipped with different anodes) are shown in Figure 3 a. The power density of the RVC bioanode reached 59.7 mW m −2 , which was 4.7 and 1.6 times higher than those of the NF (37.1 mW m −2 ) and CF (12.6 mW m −2 ) bioanodes, respectively. To further elucidate the role of the RVC anode in enhancing the power-generating performance of the MFC, electrochemical impedance spectroscopy (EIS) measurements were performed (Figure 3b). The total resistance of the MFC was the sum of the ohmic resistance and charge-transfer resistance (R ct ). R s and R ct represented the anolyte and charge-transfer resistances, respectively. The R ct of the RVC-MFC (X) was 99.9% and 99.5% lower than those of NF-MFC and CF-MFC, respectively, indicating a stronger interface interaction between the RVC surface and the biofilm, as well as faster extracellular electron transfer on the biofilm surface. The electrochemical behavior of the bioanodes was analyzed using cyclic voltammetry (CV) measurements to characterize the electrocatalytic activity of electroactive bacteria on the anodes (Figure 3c). The anode exhibited a high current response after enrichment with electroactive bacteria, confirming that the electroactive biofilms played a crucial role in influencing the MFC performance. Compared to other bioanodes, the larger CV curve area of the RVC bioanode suggested the presence of more active sites on its surface, which enhanced extracellular electron transfer and power generation in the MFC. Moreover, no significant redox peaks were observed in the CV curve of the RVC bioanode, suggesting that almost no redox mediators were generated. This indicated that direct electron transfer was the major extracellular electron transfer pathway through which microorganisms acted on the RVC anode. In a differential pulse voltammetry (DPV) curve, the peak of the current curve in the RVC-MFC was higher than those of the other two anodes (Figure 3d), as shown by the red dashed component, indicating that RVC had better electrical conductivity. 2.4. Anodic Biofilm Spatial Distribution and Microbial Communities The biofilm morphologies of the NF, CF, and RVC anodes are displayed in Figures 4 a–c. Bacteria were not uniformly layered on the surface but clustered as clumps attached to the skeletons of the NF and RVC and to the fibers of the CF. In addition, at low magnification, more clusters were present on the RVC anode than on NF and CF anodes, and the microorganisms on the three anodes were mostly spherical and rod-shaped, but the bacteria on the CF surface were arranged tightly, and impurities were more abundant on the NF anode. Further monitoring of the spatial distribution of bacteria, performed using Confocal Laser Scanning Microscope‌ (Figures 4d–f), revealed that 84% and 88% of bacteria on the NF and RVC surfaces were still alive after long-term operation, respectively, but only 54% of bacteria maintained activity on the CF anode. The living bacteria almost entirely covered the skeleton of the macroporous NF and RVC, where, for CF, live bacteria were mainly distributed at the interface between the CF and the anolyte, and the dead bacteria were mostly found within the CF. This was directly related to the dense structure of the CF and, thus, resulted in difficulty in biofilm renewal. For CF, the pore type, formed by the intersection of carbon fibers, had a more negative impact on the mass transfer of CB, compared with macroporous NF and RVC; on the other hand, the diffusion of the produced chloride ions was restricted, resulting in slower CB removal. Phylums of Proteobacteria and Bacteroidetes were predominant among the three anodes, with combined proportions of >93.1% ( Figure 5 a); these phylums are common CB degraders, [54,55,56] and a remarkable aromatic-compound user (Actinobacteria) was 1.81−2.54 times more abundant in the RVC biofilm than in the CF and NF biofilms, [54,57,58] partly accounting for the rapid CB degradation in MFC-RVC. At the genus level, as shown in Figure 5b, the bacteria with the highest proportion in biofilms was Comamonadaceae , with values of 26.3%, 21.1%, and 22.8% for MFC-RVC, MFC-CF, and MFC-NF, respectively, followed by Pandoraea sp., which accounted for over 10% in all MFCs. Comamonas species exhibited proportions 7.2%, 5.5%, and 5.9%, respectively. Comamonadaceae and Comamonas are typical exoelectrogens, capable of performing VOC oxidization and power generation [59] . For example, Cai et al. found that Comamonas could perform Cl-VOC dechlorination, [60] while both Comamonadaceae and Comamonas potentially synergized to promote electron transfer between an anode and biofilm. [61] Delftia sp. and Chitinophagaceae sp., with proportions ranging from 2% to 5%, were sensitive to the metal-containing NF and RVC anodes, while Chryseobacterium sp. and Flavobacteriaceae sp. preferentially adhered to the carbon-containing anode. In the RVC containing both metal and carbon, Mesorhizobium sp., Rhodopseudomonas sp., and Microbacteriaceae sp. were unique, with many of these species potentially playing important regulatory roles in the high performance of the MFC-RVC. Overall, the microflora on the carbon- and metal-based MFCs somewhat differed owing to the toxic effect of the metal; electrochemically active species were dominant on the NF and RVC electrodes, and degrading bacteria had larger proportions on the RVC and CF anodes. 2.5. Anodic Biofilm and Metabolic Activities The biomass on the anode and in the anolyte were quantified based on the Optical Density at 600nm (OD 600 ),which can indirectly measure the bacterial density. First, the anode was cut and shaken for 30 min for OD 600 detection. As shown in Figure 6 a, the anodic and anolyte biomass both increased with increases in the CB concentration from 50 mg L −1 to 100 and 150 mg L −1 in the three MFCs, corresponding to an increased CB removal rate. When 200-mg L −1 was added, the biomass on the three anodes and in the anolyte decreased, causing a decline in CB removal, as shown in Figure 2b. The output voltage shown in Figure 2a exhibited the same trends as biomass, indicating that the excess CB was toxic for both degraders and exoelectrogens in terms of cell growth and viability. Of note, the biomass on the RVC anode was significantly higher than that of NF and CF anodes at both CB concentrations, and the biomass in the MFC-RVC anolyte was the lowest among the three MFCs. The OD 600 of the anode oscillation ranged from 0.649 to 2.680, which was 43~113 times higher than that in the anolyte (0.0039~0.0295), confirming that the performances of the MFCs were directly related to the growth on the anode while in the anolyte. Specifically, when the CB concentration was 150 mg L −1 , the OD 600 of the RVC oscillation reached 2.680, which was 31.2% and 110% higher than those of NF (2.043) and CF (1.279), respectively, reflecting its outstanding biocompatibility with microbial communities. The extracellular polymeric substance (EPS) secretion of the anodic biofilm was further extracted for enzyme protein (PN) and polysaccharide (PS) analyses, which revealed the metabolic level and adherence capacity of the microorganisms respectively. [62] The PN/PS ratio represented the adaption ability of the biofilm, [63] with both PN and PS content related the anodic biomass. [64] As shown in Figure 6b, when 50-mg L −1 CB was added, the content of PS in the three biofilms was 1.50−1.86 times higher than that of PN, while it was 1.11−1.49 times lower when the CB concentration was increased to 150 mg L −1 . Moreover, at 150-mg L −1 CB, the PN/PS values were 0.82, 1.25, and 1.76 for the NF, CF and RVC biofilms, respectively, which were 1.53, 1.88, and 2.64 times higher than those at a CB concentration of 50 mg L −1 . This was because the MFCs were start-up at a low CB concentration (50 mg L −1 ). During the initial period, microorganisms needed to secrete a large amount of PS to help them adhere to electrodes and rapidly form biofilms, while, during the stable operation period (150-mg L −1 CB), the biofilm possessed better adaptability and the carbon source was mostly converted to biomass for CB removal and power generation, which is consistent with the results shown in Figures 2 and 6a. During stable operation of the three anodes, the RVC biofilm showed the highest PN secretion level and PN/PS ratio (1.76), the latter of which was 1.15% and 41.1% higher than those of the NF (0.82) and CF (1.25) biofilms, further confirming the superiority of the carbon–metal-composite anodes. Dehydrogenase (DHO) and cytochrome C (Cyt-C) played important roles in microbial metabolism; CB was oxidized by DHO and then, through Cyt-C, released the energy required for use by cells, and higher levels of DHO and Cyt-C corresponded with higher microorganism activity. [65,66] As shown in Figures 6 c and d, at a 150-mg L −1 CB concentration, the DHO activity and Cyt-C content of the three anodic biofilms were higher than those at 50 mg L −1 . In addition, the DHO of the RVC biofilm reached the highest at 150-mg L −1 CB, with a value of 10.80 U g −1 , which was 3.8 and 1.3 times higher than those of the CF (2.83 U g −1 ) and NF (7.75 U g −1 ) biofilms, respectively. Meanwhile, the Cyt-C content reached 105.13 nmol L −1 , which was 37.3% and 7.4% higher than those of NF (76.60 nmol L −1 ) and CF (97.93 nmol L −1 ), respectively. This indicated that, at a high CB concentration, more chemical energy stored in CB was converted to electricity in the MFC-RVC and that more energy was provided for cell growth, which greatly corresponds to the results shown in Figures 6a and 3a. On the other hand, Cyt-C is frequently reported because of its effectiveness in performing direct electron transfer [67] , and the prominent electrochemical activity of the RVC biofilm, as shown in Figure 3, was closely related to its high Cyt-C content. 2.6. Products in CB Degradation CO 2 and Cl - were the major end products in CB degradation, as shown in Figures 7 a and 7b, with the CO 2 produced in the three MFCs being positively correlated with CB concentration, whereas mineralization was negatively correlated. This was because, with increasing CB concentration, more CB was converted to biomass. The dechlorination efficiency of CB in all three MFCs exceeded 65%, and it even exceeded 85% at 100-mg L −1 CB, significantly surpassing that of other biosystems. [68] The MFC equipped with an RVC anode showed higher CO 2 production, mineralization, and dechlorination than the other two MFCs, and the differences among the three MFCs were most evident at 150-mg L −1 CB. The MFC-RVC generated 29.4 g m −3 of CO 2 , which was 48.3% and 21.4% higher than those of the MFC-CF (19.8 g m −3 ) and MFC-NF (24.3 g m −3 ); the mineralization rate of 12.6% of the MFC-RVC was 48.4% and 21.3% higher than those of the MFC-CF and MFC-NF; the dechlorination rate of the RVC biofilm was 95.6%, which was 36.7% and 7.7% higher than those of the CF (69.9%) and NF (88.8%) biofilms respectively; according to analysis of the microbial communities, the dominant benzene and Cl-VOCs were utilized by Comamonadaceae sp., Comamonas sp. , Proteobacteria sp., and Actinobacteria sp., resulting in improved mineralization and dechlorination of CB in the MFC-RVC, as well as the Microbacteriaceae and other trace genera. It has been reported that CB first combines with water molecules via the tricarboxylic acid cycle to produce chlorophenols, then with chlorobenzenediols, [21,52] and further with CO 2 and Cl - via glycolysis. To further investigate the reason for the low CB degradation rate observed at 200 mg L −1 , the organic matter within cells was broken down and detected via 3D fluorescence. As shown in Figure 7, the presence of numerous fluorescence peaks indicated more types of metabolites, and the intensity of the fluorescence peaks indicated a higher content of metabolites. [69] A horizontal comparison of Figures 7c–e and 7f–h showed that the RVC-MFC produced more types metabolites than the CF-MFC and NF-MFC, while a vertical comparison revealed that the content of metabolites at 100-mg L −1 CB was higher than that at 200-mg L −1 CB (Figures 7c and f, as well as Figures d and g or e and h). This indicated weakened microbial activity and an inhibitory effect on CB-degrading bacteria at a 200-mg L −1 concentration, which is consistent with the DHO activity. 2.7. Mechanism Analysis The mechanism of CB removal by the 3D macroporous MFC electrode is shown in Figure 8 . A larger pore size (>100 μm) in the three anodes provided substantial space for microorganism colonization, resulting in the formation of a thicker biofilm compared to that of 2D electrodes. On the other hand, the rapid transfer of CB and the generated products (such as Cl - ) in larger-pore anodes promoted the uptake of CB by microorganisms while preventing local acidification and alkalization of the electrolyte. NF exhibited much higher conductivity than that of CF, resulting in an accelerated transfer of electrons between the anode and microorganisms, and some nickel may have participated in the catalytic oxidation of CB, thus improving the efficiency of CB degradation and reducing the charge-transfer resistance at the anode interface. CF was more biocompatible than NF, which was conducive to the adhesion of microorganisms. In the carbon–nickel RVC anode, the high conductivity of Ni and the biophilicity of carbon were retained and, thus, resulted in the highest current output and CB degradation rate. 3. Conclusion The performances of three 3D anodes with pore sizes greater than 0.5 mm were compared, and the carbon–metal-based RVC anode exhibited the most conductivity, hydrophilicity, and biocompatibility, as demonstrated by the output voltage, power density, contact angle, XRD pattern, and biomass. EPSs, DHO activity, and Cyt-C confirmed that the biofilm activity of MFC-RVC was higher than those of MFC-NF and MFC-CF. The average CB degradation rate reached 14.4 mg L −1 h −1 , corresponding to a dechlorination rate of 95.6%, and a maximum output voltage of 248.3 mV under the dominance of Comamonadaceae , Comamonas , and Microbacteriaceae . RVC bioanodes present advantages in power generation and contaminant removal; however, the high cost of RVC may limit their industrial applications. The results of this study are useful for achieving affordable NF and CF modification. 4. Experimental Section Three sets of ”H”-type double-chambered MFCs, installed using NF, CF, and RVC anodes (corresponding to MFCs defined as MFC-NF, MFC-CF, and MFC-RVC, respectively), were applied as reactors. Two Polytetrafluoroethylene cap-sealed glass cylinders, separated by a Nafion 117 proton-exchange membrane, were used as anode and cathode chambers. The dimensions of the three anodes were 20 mm × 20 mm × 3 mm, and a platinum sheet (20 mm × 20 mm × 0.5 mm) was used as the cathode in the three MFCs. The anodes were cleaned in an orderly manner using acetone, ethanol, and distilled water, and each step was repeated three times. The anode and cathode were connected by an external resistance of 1000 Ω, and Ag/AgCl (+0.197 vs. SHE) was used as the reference electrode. An activated sludge suspension obtained from Zhejiang Petrochemical Co., Ltd. (Zhoushan, China) was used as the inoculum to start-up the MFCs. Ten milliliters of bacterial suspension and 35 mL of a Phosphate Buffered Saline (PBS) solution were mixed and added to the anode chamber (total volume of 70 mL), and 50 mL of 50-mmol L -1 potassium ferricyanide containing PBS was used as the catholyte. The composition of the PBS was the same as that used in a previous study. [62] During the initial start-up period, a gradient of sodium acetate (0.5–0 g L -1 ) was added as an auxiliary carbon source to enhance the growth of exoelectrogens and CB degraders. During the late start-up stage, CB was supplied as the sole carbon source; when the removal efficiency of 50-mg L -1 CB was stabilized at 90% and the maximum output voltages were equal over two consecutive cycles, the start-up of the MFCs was completed. The degradation performance was then assessed by adding 50, 100, 150, and 200 mg L -1 of CB in batchwise mode at 30 °C. The electrochemical, chemical, and biological characteristics of the MFCs were detected simultaneously at each CB concentration. Once the output voltages of MFCs were decreased to below 50 mV, the anolyte and catholyte were refreshed simultaneously.The sodium acetate, CB, and potassium ferricyanide, all with purities ≥99.0%, were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). The anode and cathode of each MFC were connected to a recorder to monitor the output voltage (MIK R5000C; Meacon, Hangzhou, China) and the sampling interval was set at 10 min. The electrochemical activities of the three anodes, based on current density, power density, DPV, EIS, and CV results, were characterized using a rheostat or electrochemical workstation (BioLogic VSP 300, France). For the current- and power-density measurements, a circuit was first cut off for 2 h and then connected using an external resistance ranging from 100 Ω to 100 kΩ. Subsequently, the voltage was recorded for further calculation using the equation U 2 /(R·S), where U, R, and S represent the detected voltage, external resistance, and anode area, respectively. The detection parameters for DPV, EIS, and CV were consistent with those in previous studies. [62,70] The degradation of CB and the production of CO 2 were determined using a gas chromatograph (GC; Agilent 7890 B, USA). For CB detection, the GC was equipped with a flame ionization detector and HP-INNOWax column (30 m × 320 μm × 0.5 μm). The temperature of the injector, detector, and oven were 250, 280, and 200 °C, respectively, and nitrogen was used as the carrier gas at a flow rate of 21 mL min -1 . For CO 2 detection, the GC was equipped with a thermal conductivity detector and an HP-Plot-Q column (25 m × 320 μm × 10 μm). The temperature of the injector, detector, and oven were 100, 180, and 40 °C, respectively. Helium was used as carrier gas at a flow rate of 2 mL min -1 . Cl - was identified using an ion chromatograph equipped with an Ionpac AS19-HC column (ICS-2000 Dionex, USA). KOH (40 mmol L -1 ) was used as the eluent at an elution rate of 1 mL min -1 . A fluorescence spectrophotometer (RF-6000, Japan) was used to analyze CB-degraded organic products under excitation wavelengths of 220–450 nm, emission wavelengths of 220–550 nm, a scanning speed of 2000 nm min -1 , and excitation and emission band widths of 10 nm. The hydrophobicity of the three anodes was measured using a contact-angle meter (SZ-CAMB3; Shanghai Xuanjun Instruments, Shanghai, China). Meanwhile, the resistivities of the anodes were determined using a resistivity instrument (HPS2663; Helper Electronics Technology, Changzhou, China). The crystallinities of the anode materials were determined using XRD (MiniFlex 600, Japan) at 40 kV, a tube current of 40 mA, and a Cu-target X-ray source ranging from 10° to 70° over intervals of 0.02°. The biomass in the anolyte were charactered based on the OD 600 value. For anodic attached microorganism, cut anode samples were shaken and sonicated at 40 kHz for 30 min and then measured using a UV spectrophotometer (Cary 60 UV-Vis, Agilent, USA) at a wavelength of 600 nm. The secretion of EPSs was determined as previously described [62] and quantified via heat treatment and an anthracene–sulfuric acid colorimetric assay. [71] The PN content in the EPSs was determined using a bicinchoninic acid assay PN-concentration assay kit (Phygene, Fuzhou, China), while the PS content in the EPSs was determined using a total-PS-content assay kit (Isejyu Bio, Lianyungang, China). The microstructural morphologies of the bare anode surfaces and biofilms were observed using field-emission scanning electron microscopy (SU8010; HITACHI, Japan). For biofilm observation, the three anodes (NF, CF, and RVC) were first immersed in 2.5% glutaraldehyde, fixed with 1% osmium acid, and then observed after gradient ethanol dehydration, vacuum drying, and gold plating. The observation of live/dead bacteria and their distributions on the anodes were performed via CLSM (LSM 780, Zeiss, Germany). Freshly collected samples were first stained using a mixture (1:1) of SYTO-9 and propidium iodide (BacLight Bacterial Viability KL-7007, Canada) for 20 min under dark conditions, after which they were then rinsed twice using 50-mmol L -1 PBS. Over intervals of 2 nm, the excitation wavelength of SYTO9 was 488 nm/503 nm, while that of propidium iodide was 543 nm/503 nm. Finally, the scanned images were composited and exported using the ZEN software, and calculation of the proportions of live bacteria was conducted using ImageJ. The microbial community composition of the anodic biofilm was analyzed using high-throughput sequencing. 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Montilla, J. Phys. Chem. C 2017 . 121, 1587. [68] T. Li, H. Li, C. Li, Chem. 2020 , 250, 126338. [69] W. Li, X. Li, C. Han, L. Gao, H. Wu, M. Li, Sci. Total Environ. 2023 , 855, 158963. [70] G. Wu, H. Bao, Z. Xia, B. Yang, L. Lei, Z. Li, C. Liu, J. Power Sources 2018 , 384, 86. [71] M. F. R. Mizan, I. K. Jahid, M. Kim, K. H. Lee, T. J. Kim, S. D. Ha, Biofouling 2016, 32, 497. Supplementary Material File (figure.docx) Download 27.61 MB Information & Authors Information Version history V1 Version 1 07 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords chlorobenzene removal mechanisms microbial fuel cells microporous anodes Authors Affiliations JuP YOU 0000-0003-2714-5368 [email protected] Zhejiang Ocean University View all articles by this author Metrics & Citations Metrics Article Usage 160 views 116 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation JuP YOU. 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