Enhancing extracellular electron transfer through selective enrichment of Geobacter with Fe@CN modified carbon-based anode in microbial fuel cells

doi:10.21203/rs.3.rs-2072655/v1

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Enhancing extracellular electron transfer through selective enrichment of Geobacter with Fe@CN modified carbon-based anode in microbial fuel cells

https://doi.org/10.21203/rs.3.rs-2072655/v1

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Microbial fuel cells (MFCs) have been demonstrated as a renewable energy strategy to efficiently recover chemical energy stored in wastewater into clean electricity by exoelectrogens through a combination of microbial metabolism and electrochemical processes, yet the limited power density limits their application. Here, Fe coped carbon and nitrogen (Fe@CN) nano-material was synthesized by direct pyrolysis of FeCl₃ and urea, which was further decorated to fabricate Fe@CN modified carbon paper anode to improve the bioelectrochemical performance of MFCs. With a higher electrochemically active surface area, the Fe@CN modified anode was beneficial for enhancing microorganisms adhesion and extracellular electron transfer (EET). Accordingly, the MFC with Fe@CN modified anode (MFC-Fe@CN) successfully achieved a highest voltage output of 792.76 mV and a prolonged stable voltage output of 300 h. Interestingly, Geobacter species (15.41% abundance) was significantly enriched in the EABs of MFC-Fe@CN, while MFC-CP (unmodified carbon paper anode) favored the other exoelectrogens (Dysgonomonas and Comamonas). This work demonstrated the feasibility of development on anode catalysts for the elaboration of the catalytic principle about interface modification, which may contribute to the practical application of MFC in energy generation and wastewater treatment.

microbial fuel cells

Fe coped carbon and nitrogen

electroactive biofilms

electrochemically active surface area

extracellular electron transfer

In the past decades, over-exploitation and over-consumption of fossil fuels has created serious threats for human being, such as global warming, environment pollution, and energy shortage. Under this circumstance, bioelectrochemical systems (BESs) have shed light on the research and development of renewable energy sources due to their versatility for degradation of organic pollutant and production of green-energy simultaneous by the metabolism of exoelectrogens (such as Geobacter and Shewanella) (Sreelekshmy et al. 2020, Wang et al. 2020). As a typical BESs, however, previous researchers have demonstrated that the low efficiency of extracellular electron transfer (EET) and low power density were the obstacles for the commercial application of microbial fuel cells (MFCs) since the produced power from single MFC was too low to be used even in low consumption devices (Song et al. 2016, Yong et al. 2014a).

Commonly, exoelectrogens in the anode chambers tend to aggregate on anodes and evolve into electroactive biofilms (EABs), which are the core component of MFCs for degradation of organics and generation of electrons (Wu et al. 2020, Yong et al. 2014b). EABs are similar to natural biofilms in the spatio-temporal formation, composition, and structure but differ in their electrochemical properties since EABs act as electrons sink and transfer electrons to electrode. Undoubtedly, the formation process of EABs was proved to be affected by the anode properties, such as the interfacial area (Sreelekshmy et al. 2020), hydrophobicity (Yang et al. 2017), and biocompatibility (Li et al. 2019b), which significantly affected the interfacial catalytic interaction between EABs and electrode.

It’s also well known that the performance of MFCs relies heavily on EET between the intracellular respiratory chains of exoelectrogens and electron acceptors. When the electron acceptors (such as abiotic solid electrode) cannot enter the interior of the cells through cell membrane, the exoelectrogens transfer the generated electrons to outer-membrane conductive proteins through respiratory chain, and finally to electrodes through the direct (external c-type cytochromes or flagella) or indirect pathways (electron mediators) (Hu et al. 2019, Liu et al. 2020a).

Thus far, it is reported that the morphological structure and the surface chemistry properties of electrode used in MFCs directly influence the EET capability between anode and exoelectrogens. Different porous biomass materials (corn stems, commercial bread, and loofah sponges) were used as precursors to prepare three-dimensional (3D) electrodes with good conductivity (Mei et al. 2020). As a result, the macroporous structure in the carbonized electrodes were preferable for improving the electrochemically active surface area (EASA) and forming EABs compared with that of plain carbon felt electrode. Furthermore, a 3D microporous nitrogen-enriched graphitic carbon electrode was fabricated through facile pyrolysis and embedded in MFCs, which achieved a highest power density of 750 mW/m² (2.07-fold higher than that of control group) (You et al. 2017). These works demonstrated that the types and properties of anodes heavily determined the electron-producing capacity of EABs and EET rate accordingly, thus further affected the bioelectrochemical performance of MFCs.

To date, considering the cost of all BESs systems and experiment processes, the main types of anodes used in MFCs are carbon-based materials, such as carbon felt (Li et al. 2021a), carbon cloth (Li et al. 2021b), carbon fiber brusher (Hari et al. 2016), graphite plate (Yang et al. 2019), and so on. Nevertheless, the abovementioned anodes does not show extra breaks in the performances of MFCs, particularly since their hydrophobicity and low surface area are unsuitable for bacteria adhesion and high internal resistance unfavorable for EET (Liu et al. 2020b).

The modification and upgradation of electrodes is an attractive and effective strategy to strengthen anodic biocompatibility, enhance EET efficiency, and improve performance of MFCs (Li et al. 2020a, Yang et al. 2020b). Recently, conductive substrates (Xu et al. 2022), functional groups (Li et al. 2020a), and transition metal carbides and nitrides (Yang et al. 2020b) were consecutively employed to modify the anodes by surface modification. Researchers have found that specific metals doped carbon and/or nitrogen can significantly facilitate the EET process of exoelectrogens. Song et al. synthesized a 3D anode through combination of carbon nanotubes and Fe₃O₄ nanospheres into microporous graphene foams to fabricate a MFC system with a higher power output of 782 A/m³ (11-fold higher than that of control group) and a stronger stability (Song et al. 2016). The high affinity of Fe₃O₄ nanospheres toward exoelectrogens and the smoother EET pathway paved by microporous graphene foams were mainly responsible for the high-performance of MFCs compared with that of graphite rod anode. Wang et al. also reported that the FeS₂ nanoparticles decorated graphene anode not only enhanced the adhesion of exoelectrogens but also shorten the start-up of MFCs by enhancing the EET process (Wang et al. 2018). Moreover, Wu et al. reported that the N-doped carbon nanotubes/reduced graphene oxide (rGO) promoted the formation of EABs on anode and further achieved a maximum power density of 1137 mW/m² (Wu et al. 2018). In a word, the enhanced performances by the modification of anodes could be attributed to the following reasons: 1) the 3D structure and porosity was beneficial for exoelectrogens habitation and EABs formation (Song et al. 2016, You et al. 2017). 2) the enrichment of exoelectrogens produced more exoelectrons and achieve higher voltage output (Li et al. 2021c). 3) the lower charge transfer resistance avoided the loss of electron in EET and improved the coulomb efficiency of MFCs (Yang et al. 2021, Yu et al. 2018).

Although most works about electrode modification with metal or metal oxide integrated with some conductive materials were reported (Wang et al. 2022), few studies explored the effect of co-decoration of anodes with metal (Fe, Mn, Co, and so on) and non-metal (C, S and N) on the power performance of BESs. In this context, the present study highlights the possibility of a Fe@CN nanospheres catalyst coating on the carbon paper anode for achieving a high bioelectrochemical performance of MFCs, and the reasons for the enhanced performance were also elaborated from both biological and electrochemical perspectives. As shown in Scheme 1, Fe@CN nanospheres were synthesized through one-pot pyrolysis and further coated on the surface of carbon paper. The obtained anode had porous structure and achieved a maximum voltage output of 792.76 mV, which could be attributed to the enrichment of exoelectrogens (Geobacter) and the variation of EET pathway. This work shed new insights on fabricating efficient anodes for selectively enriching exoelectrogens and efficiently enhancing the performance of MFCs.

One-pot method for the synthesis of Fe@CN nanospheres

The Fe@CN nanospheres was synthesized by one-pot pyrolysis of Fe (Ⅲ) and carbon-nitrogen precursors. In detail, 0.5 g of FeCl₃ and 3 g of urea were dispersed in ultrapure water (10 mL), which was stirred for 20 min and then sonicated under 30°C for 60 min to obtain a homogenous solution. Next, the suspension was placed into a muffle furnace and annealed at 550°C for 2 h with a heating rate of 10°C min^− 1. The samples were denoted as Fe@CN1. Accordingly, Fe@CN0 (pure FeCl₃ without urea) was prepared in a similar synthesis process.

Characterization Of Catalyst

The microstructure detection of Fe@CN nanospheres was conducted by scanning electron microscope (SEM, Regulus-8100) coupled with an energy dispersive spectrometer (EDS). X-ray diffraction (XRD, D8 Advance) patterns of the catalyst were obtained with 2θ range of 5–90°. The Raman images were obtained using Raman spectroscopy (RAM ΙΙ). The X-ray photoelectron spectroscopy (XPS, AXIS) was employed to measure the chemical composition and atomic valence of the synthesized catalysts.

Electrode Fabrication And Mfcs Setup

Carbon paper was used as plain template for coating the prepared Fe@CN catalyst. In detail, 5 mg of powdered catalyst was dispersed in 20 µL ultrapure water, which was then mixed with 10 µL Nafion solution to prepare catalyst ink. The surface of a carbon paper (2.0 × 1.0 cm) was coated with the abovementioned catalyst ink and dried under 60°C for 2 h. The modified electrode was assembled into MFCs as the anode in the following studies.

The typical double-chamber MFCs device with 50 mL of each identical volume, separated by a proton exchange membrane (Dupont, USA), was constructed as our previous reports (Cheng et al. 2022). The anodes were the control carbon paper electrode or the above prepared Fe@CN electrode. The cathode utilized in this work was carbon paper electrode. The anode and cathode were connected with 1,000 Ω external resistance to form a closed circuit. The distance between the two electrodes was about 8 cm. The anolyte was consisted of wastewater (sampled from Nanjing wastewater treatment plant, Nanjing, China) and phosphate buffer solution (v/v : 1/4): NaH₂PO₄, 2.54 g/L; Na₂HPO₄, 4.09 g/L; NH₄Cl, 0.31 g/L; KCl, 0.13 g/L, along with minerals and vitamins solutions (Li et al. 2019a). Sodium acetate (1.28 g/L) was added into anode chamber as electron donors, and the catholyte was 50 mM potassium ferricyanide (K₃[Fe(CN)₆]) with phosphate buffer solution. During the running process, MFCs were accessed to the data collectors to record the voltage output, and the temperature in all process was maintained at 30°C. All experiments were set up in triplicate.

Characterization And Analytical Measurements

The biomass density of EABs was measured using BCA Protein Assay Kit (Li et al. 2021a). For the micromorphology detection of EABs, electrodes with EABs were extracted from MFCs and subsequently cut into small pieces. After fixed in 2.5% glutaraldehyde for 2 h, the biofilm pieces were dehydrated using a graded ethanol series (25, 50, 70, 80, 90, and 100%). Finally, the sample were dried at room temperature for 12 h and analyzed through scanning electron microscopy (SEM) (Li et al. 2021a). Extracellular polymeric substances (EPS) fractions from the EABs were extracted following the previous report (Li et al. 2019a). Polysaccharide and protein from the EPS were determined by the sulfuric acid-phenol method and BCA Protein Assay Kit, respectively (Cheng et al. 2022).

The electrochemically active surface area (EASA) was obtained through cyclic voltammograms (CV) from − 0.4 to 0.7 V with a scan rate of 10 mV/s in the electrolyte with 0.1 M Na₂SO₄ and 10 mM K₃[Fe(CN)₆] (Xu et al. 2022). The electrochemical active surface area (EASA) of prepared electrode was calculated using Matsuda’ equation:

$$\text{I}\text{p}=0.4464\text{*}{10}^{-3}*{n}^{\frac{3}{2}}*{F}^{\frac{3}{2}}*A*{\left(RT\right)}^{-\frac{1}{2}}*{D}^{\frac{1}{2}}*{C}_{0}{v}^{\frac{1}{2}}$$

where Ip was the peak current; n = 1 was the number of the transferred electrons; F = 96,487 C/mol was the Faraday’ constant; R = 8.314 J/(mol·K) was the gas constant; T = 303 K was the temperature; A represented the EASA of the prepared electrode; D = 5.79*10^− 6 cm²/s was the diffusion coefficient of the K₃[Fe(CN)₆ solution.; C₀ = 10 mM was the concentration of the K₃[Fe(CN)₆. v = 10 mV/s was the scan rate.

The polarization curves were conducted when the MFCs were in steady state with a scan rate of 10 mV/s from − 0.8 to 0 V. Cyclic voltammograms were measured on a three-electrode configuration with a reference electrode (Ag/AgCl), a working electrode (the EAB anodes), and a counter electrode (carbon paper electrode) from − 0.8 to 0.8 V at a scan rate of 10 mV/s.

Characterization of Fe@CN catalysts

The dispersed iron anchored C-N supporter was prepared by one-pot pyrolysis of FeCl₃ and urea (Scheme 1), which was named as Fe@CN catalysts.

SEM images (Fig. 1) revealed that these Fe@CN samples obtained at 550℃ from the precursor with different mass ratio of FeCl₃/urea showed significant different surface morphology. It was noteworthy that Fe@CN0 exhibited an irregular geometric configuration consisting of a smooth sphere and blocky structure, which was resulted from the interaction of iron and oxygen during pyrolysis. The corresponding elemental mapping images demonstrated that C, N, O and Fe elements relatively uniform distributed Fe@CN1 sample (Fig. 1c-f), which implied that Fe was successfully doped with C and N element.

XRD patterns of the prepared Fe@CN_x catalysts were shown in Fig. S1a. The peaks in Fe-CN0 at 24.2°, 33.2°, 35.7°, 40.9°, 49.5°, 54.3°, 57.7°, 62.7°, and 64.1° matched well with the (0 1 2), (1 0 4), (1 1 0), (1 1 3), (0 2 4), (1 1 6), (0 1 8), (2 1 4), (3 0 0) crystal facets of Fe₂O₃ (JCPDS No. 33–0664) (Li et al. 2022). The characteristic diffraction peaks in Fe@CN0 disappeared when Fe doped into urea to form Fe@CN1, which suggested the effective recombination of carbon and nitrogen structure. Moreover, Raman spectra was recorded to further reveal the defect sites and disordered structure of Fe@CN catalysts. The D-band was induced by the vacancies and heteroatom doping while the G-band was related to the in-plane stretching vibration of sp2 hybrid atoms (Yang et al. 2020a). As shown in Fig. S1b, the ratio of I_D/I_G in Fe@CN1 catalyst was 1.23, indicating that the existence of more defect sites (Liu et al. 2020b).

The surface chemical composition and valance states of these catalysts were also scrutinized by X-ray photoelectron spectroscopy (XPS). The C1s spectra of Fe@CN1 revealed the presence of C-C (284.78 eV), C-N (286.48 eV), and N-C = N (289.18 eV) functional groups on the surface of Fe@CN1 (Fig. 2a). Moreover, the deconvoluted N1s spectrum of Fe@CN1 exhibited two peaks at 400.38 and 403.38 eV, which were attributed to pyrrolic N and graphitic N, respectively (Fig. 2b). Previous study reported that graphitic N could enhance the material biocompatibility and reduced the start-up time of MFCs (Li et al. 2020a, Zhao et al. 2019). The pyrrolic N was responsible for improving the power density of MFCs systems (Liu et al. 2014, Xie et al. 2017). The deconvoluted Fe2p spectra of Fe@CN1 was presented in Fig. 2c, which was assigned to Fe^2+/3+2p_1/2, Fe^2+/3+2p_3/2, and their satellites. In details, the peaks at 711.87, 714.52, and 718.90 eV in the Fe2p spectrum were assigned to Fe²⁺2p_3/2, Fe³⁺2p_3/2, and their satellites, while Fe²⁺2p_1/2, Fe³⁺2p_1/2, and their satellite peaks appeared at 725.42, 729.38, and 732.90 eV, respectively. Thus, the transition of solid-state redox couples Fe³⁺/Fe²⁺ was expected to be beneficial for electron transfer (Li et al. 2015, Rethinasabapathy et al. 2021).

Electrochemical Active Surface Area Of Different Anodes

To investigate the electrochemical performance of the modified electrode, EASA test was conducted in a three-electrode system with prepared Fe@CN modified electrodes, titanium wire, and Ag/AgCl electrode as working electrode, counter electrode, and reference electrode, respectively. Previous studies proved that EASA represented the actual electroactive area on electrode and a higher EASA facilitated the adhesion of electrochemical active bacteria and benefited the bioelectrochemical performance of MFCs ultimately (Chen et al. 2020, Xu et al. 2022).

Additionally, similar current curves with a pair of reversible redox peak were displayed in Fig. S2, which indicated that the chemical reduction reaction was not changed in this system. However, the catalytic curve of the blank electrode (carbon paper without modification) delivered a Ip value of 0.98 mA with a corresponding EASA of 1.53 cm² in K₃[Fe(CN)₆]. Distinctly, the peak current values of the electrodes with Fe dopant improved significantly. To be more specific, the highest current value was increased to 1.14 mA with lager EASA of 1.78 cm², and further increased to 2.35 cm² (Fig. S2). The fact also indicated that Fe@CN catalyst was successfully coated onto the surface of carbon paper, which provided more electrochemical active sites for capturing electrons from exoelectrogens.

Bioelectrochemical Performances Of Mfcs With Fe@cn Modified Anode

The MFCs initiated with mixed-culture were inoculated with wastewater as electron donors and loaded with a 1000 Ω external resistance. The power performance of all the launched MFCs showed a similar trend in the initial 4-running cycles, which increased steadily after replacing with fresh anolyte (Fig. 3). In particular, the voltage output of MFC-CP increased and achieved the maximum of 616.39 mV at about 100 h, which indicated that the exoelectrogens evolved to form matured EABs and started to produce robust electrons. In contrast, MFC-Fe-CK and MFC-Fe@CN achieved higher voltage outputs of 711.89 mV and 792.76 mV without long start-up periods, respectively (especially in MFC-Fe@CN), which also maintained for a long time (e.g. over 300 h in the 1st cycle). Furthermore, the voltage output of MFC-CP declined quickly after reaching the maximum voltage. The maximum voltage output of MFC-CK with the Fe@CN2 modified anode, however, was not enhanced (573.48 mV) and the start-up time (about 100 h) was also not accelerated compared with that of MFC-CP (plain carbon paper anode), which indicated that the increased urea proportion was not the primary cause for the enhanced electrochemical performance of anodes (Fig. S4). Compared with other MFCs, the maximum voltage output of MFC-Fe@CN could be resulted from its large EASA, which was also reported in previous studies. You et al. prepared a 3D microporous nitrogen-enriched graphic carbon electrode for efficient bioelectricity generation in MFCs (You et al. 2017), which showed a higher EASA (109.01 cm²) with higher voltage output (508.00 mV) compared with that of carbon cloth anodes (EASA of 31.23 cm² with voltage output of 395.00 mV).

Interestedly, on the other hand, MFC-Fe@CN possessed a stable voltage output during each period. In details, the voltage outputs of MFC-Fe@CN were 706.79, 718.14, 722.42, and 682.54 mV at the end of the 1st to 4st cycle, respectively, while the voltage outputs of MFC-CP decreased to about 0 mV in each cycle (0.11, 5.82, 26.19, 4.87 mV for the end of the 1st to 4st cycle, respectively). The long stable voltage output was the precondition for the real application of MFCs, which potentially indicated the superiority of the prepared anodes in the present study.

To investigate the electrochemical activity of EABs on different modified anodes, CV measurements under turnover condition were employed to test the redox state of the matured EABs at a scan rate from 10 to 200 mV/s. The CV spectrum (Fig. 4) showed that various redox peaks appeared, which might be due to the altered EET efficiency of EABs evolved on each modified anode. Particularly, there was only a pair of redox peaks of the anode in MFC-CP, with the peak center at -0.31 V and a peak current of 0.23 mA. While the CV spectrum of the anodes modified with Fe@CN1 and Fe@CN2 in MFC-Fe-CK and MFC-Fe@CN showed the similar trend, respectively, which implied that the same redox substrates were responsible for EET in these two systems. In addition, the peak at -0.12 V almost disappeared in MFC-Fe-CK, which, in contrast, possessed 0.45 mA of catalytic current in MFC-Fe@CN. Moreover, the other peaks appeared similar redox potential (0.32 V in MFC-Fe-CK, 0.30 V in MFC-Fe@CN) with the different catalytic current (0.58 mA in MFC-Fe-CK, 0.97 mA in MFC-Fe@CN) indicated that the EABs in these two MFCs had significant different catalytic activities. The abovementioned results demonstrated that the Fe@CN modified electrodes increased electrocatalytic activity toward the oxidation of acetate of exoelectrogens, which were in good accordance with the voltage outputs (Fig. 3). When the scan rate increased from 10 to 200 mV/s, the peak current also increased consequently. Interestingly, the peak currents of all MFCs exhibited a good line relation with the scan rates, which suggested that the electrochemical reactions on all anodes were the typical surface-controlled processes (Li et al. 2020b).

The polarization and power density curves of the MFCs with different modified anodes were also tested with LSV. The maximum power density of MFC-Fe@CN was 0.434 W/m², which was 13.91%, and 183.66% higher than that of MFC-Fe-CK (0.381 W/m²) and MFC-CP (0.153 W/m²), respectively (Fig. 5b). The similar open-circuit voltages were exhibited in MFC-Fe-CK (668.18 mV) and MFC-Fe@CN (655.98 mV), which were also 39.49% and 34.55% higher than that of MFC-CP (487.52 mV), respectively. These results indicated that the Fe@CN modified carbon paper anodes were more suitable for the attachment of exoelectrogens (Wang et al. 2018).

EIS experiment was also performed on all MFCs to identify the electron transfer resistance between exoelectrogens and anode (Fig. 5b). The similar solution resistance (Rs) was obtained (Table. S1), which indicated that the electrodes modified with Fe@CN had no impact on Rs. Apparently, the value of charge transfer resistance (Rct) in MFC-Fe-CK (29.59 Ω) and MFC-Fe@CN (26.54 Ω) were greatly lower than that of in MFC-CP (36.24 Ω), suggesting that the modified anode decreased Rct. As discussed in our recent report, lower Rct could result in a faster extracellular electron transfer rate (Cheng et al. 2022). These results implied that Fe@CN modified anodes in MFC-Fe-CK and MFC-Fe@CN possessed the higher conductivity, which could be resulted from their larger EASA and more active interfacial interaction between exoelectrogens and electrode.

The Structure And Morphology Of Eabs On The Anodes Modified With Fe@cn

As previous reports, the performance of electrons generation in MFCs was related closely to the activity of EABs on anode, and the architecture of EABs was one of the key cores determining the performance of MFCs (Caizan-Juanarena et al. 2019). After over 700 h operation, stable voltage outputs were achieved in all MFCs, indicating that exoelectrogens successfully attached on the surface of modified anodes to form measurable EABs. As shown in Fig. 6, only a small part of anode was coated with exoelectrogens in MFC-CP, on which the cells’ adhesion was limited and loose due to the lower specific surface area of the smooth surface of carbon paper. On the other hand, porous and sponge-like structure was obtained after modifying with Fe@CN catalysts, which was more favorable for attracting exoelectrogen cells in wastewater from the first cycle of voltage output. Therefore, the shorter start-up time were presented in MFC-Fe@CN with the faster maturation of EABs on anodes. In addition, the three-dimensional structure of anodes in MFC-Fe-CK and MFC-Fe@CN could capture and transfer electrons from the outer-membrane of exoelectrogens in multiple directions. The SEMs results were further supported by the biomass analysis in Fig. S3a, which showed that the content of biomass in MFC-CP (4.08 mg/cm²) increased to 5.95 mg/cm² (MFC-Fe-CK) and 7.27 mg/cm² (MFC-Fe@CN), respectively.

The Modified Anodes Modulated The Eps Production Of Eabs

As an integral surrounding component of cells and/or biofilms, EPS widespread on the surface of microorganism. It’s well known that polysaccharides and proteins (the two main components), and other substrates (e.g. c-type cytochromes and humics) were responsible for the heterogeneity of EPS (Xiao et al. 2017). Polysaccharides were proved to protect cells against unfavorable environment (Cheng et al. 2022, Li et al. 2021a). During the colonization and maturation of EABs on anodes, the mixed-culture trended to produce some secretions and further to form EPS to package the biofilm from outer environment. In this study, we found that the content of proteins (0.97, 1.31, and 1.57 mg/cm² for MFC-CP, MFC-Fe-CK, and MFC-Fe@CN, respectively) in EPS of EABs was apparently higher than that of polysaccharides (0.85, 1.03, 1.12 mg/cm² for MFC-CP, MFC-Fe-CK, and MFC-Fe@CN, respectively) among all MFCs (Fig. S3b). Previous study showed that intracellular electrons generated from the exoelectrogens was hard to be transferred to the electrode due to the thicker EABs (over tens of microns) (Xiao et al. 2017). Nevertheless, proteins inlaid in EPS was considered to contain multiple redox molecules to overcome the insulativity of EABs (mainly was polysaccharides), which significantly accelerated EET together with other redox-active substrates (e.g., c-type cytochromes) (Li et al. 2021a, Xiao &Zhao 2017). In this case, a higher proteins content in EPS of the EABs on Fe@CN modified anodes was responsible for the enhanced EET efficiency, which also benefited the growth of exoelectrogens in MFCs systems with insoluble electron acceptors. In this work, to be more specific, the maximum content of protein in the EPS was 1.57 mg/cm² in MFC-Fe@CN compared with that of MFC-Fe-CK (1.31 mg/cm²), and MFC-CP (0.97 mg/cm²). On the other hand, polysaccharides played an important role to form the carbon skeleton of biofilms, although it was reported to be nonconductive in some studies (Yang et al. 2019). The similar content of polysaccharides was found in MFC-Fe@CN (1.12 mg/cm²) and MFC-Fe-CK (1.03 mg/cm²), which were significantly higher than that of MFC-CP (0.85 mg/cm²). Moreover, it was reported that the content of extracellular polysaccharides was often improved with the increasing environmental stress. Here, the Fe@CN nano materials might induce slight stress to the mixed-culture and active the production of polysaccharides. Apparently, the higher content ratio of proteins/ polysaccharides occurred in MFC-Fe-CK and MFC-Fe@CN compared with that of MFC-CP. The high content of proteins in EPS compensated the possible electrons transfer resistance caused by the poor conductivity of polysaccharides. The Fe@CN modified anodes improved the electrochemical activity of EPS by adjusting the content of EPS and the ratio between different component, and thus enhanced the EET efficiency of EABs. Overall, the production of EPS on different anodes would be finely regulated by cells to keep balance between EET efficiency and self-protection against environmental stress (Li et al. 2021a).

Microbial Community Analysis Of Eabs On Fe@cn Modified Anodes

When achieving a stable voltage output, high-throughout sequencing was performed to investigate the microbial community structure of EABs on different Fe@CN modified anodes. The dominant phyla of three EABs were all Proteobacteria, Bacteroidetes, and Firmicutes (Fig. 7). Proteobacteria were mainly Gram-negative and considered to be the most well-known exoelectrogens, Bacteroidetes were responsible for the electron generation, and Firmicutes were related to the fermentation and/or degradation of complex organic substrates (Li et al. 2019a, Li et al. 2021b). It was noted that Proteobacteria had the highest proportion with the abundances of 53.80%, 80.10%, and 55.16% for MFC-CP, MFC-Fe-CK, and MFC-Fe@CN, respectively. The relative abundances of Bacteroidetes decreased from 38.26% (MFC-CP) to 5.60% (MFC-Fe-CK) and 24.37% (MFC-Fe@CN), and Firmicutes increased from 3.65% (MFC-CP) to 11.40% (MFC-Fe-CK) and 11.48% (MFC-Fe@CN), respectively. These results implied that the microbial community of EABs changed tremendously after the carbon paper modifying with Fe@CN nano-catalysts.

Additionally, at the genus level, obvious difference on the microbial composition of all EABs were obtained, which suggested that different functional microbes enriched on different anodes. Detailedly, in MFC-CP, the microbial population was dominated by Dysgonomonas and Comamonas, which together accounted for 53.58% of the total microbial composition. These bacteria were considered to be the efficient exoelectrogens with higher power generation capacities in previous reports (Kong et al. 2022, You et al. 2021). However, the proportion of these bacteria decreased dramatically in MFC-Fe-CK (Dysgonomonas: 33.68–2.48%; Comamonas: 19.90–1.35%) and MFC-Fe@-CN (Dysgonomonas: 33.68–3.00%; Comamonas:19.90–4.40%). At the same time, Geobacter was enriched to the proportion of 10.49% and 15.41% in MFC-Fe-CK and MFC-Fe@CN, respectively. The reasons for the selective enrichment of Geobacter species in MFC-Fe-CK and MFC-Fe@CN were in multiple fronts. On the one hand, Geobacter was well-known as the most typical exoelectrogen in most studies, who had high capability of direct EET pathway via nanowire and c-type cytochromes (Ueki 2021). The two EET pathway strongly depended on the physical connection between the surface of both cells/nanowire and anode. Interestingly, the sponge-like and porous structure of the modified anodes were beneficial for the contact with Geobacter, which could be one prerequisite for the accelerated start-up time of MFC-Fe@CN. On the other hand, a higher EASA of the modified anodes provided more electroactive sites for exoelectrogens. Figure 7b showed that Comamonas and Dysgonomonas enriched in MFC-CP with more abundance while Geobacter reached a low proportion (2.54%), which demonstrated the selective enrichment of exoelectrogens on anode was largely changed by Fe@CN nanospheres. In addition, the shift EET pathway in MFC-CP, MFC-Fe-CK, and MFC-Fe@CN implied the varied main exoelectrogens responsible for electron generation, which was consistent with the dynamics of microbial community. Zhu demonstrated that the relative abundance of Geobacter increased from 10.00–33.96% when carbon cloth anode was modified with heteroatom-doped (N, P, S, Co) porous carbon nanoparticle, while Acinetobacter (also reported as an exoelectrogens) decreased from 28.24–9.00% (Zhu et al. 2022). And thus, the higher power density (1.72 W/m²) and current density (4.52 A/m²) were achieved with the modified anode compared with that of plain carbon cloth anode (0.95 W/m² of power density and 3.12 A/m² of current density). The above results also revelaed that the evolution of EABs in the composition and proportion of exoelectrogens varied greatly on different anodes with different structures and properties, which may be attributed to the variable responses of different microbial species to the Fe@CN modified anodes.

In summary, the novel Fe-coped carbon and nitrogen (Fe@CN) catalyst had been successfully synthesized by one-pot pyrolysis and decorated on carbon paper as MFCs anode. The Fe@CN nano-particles significantly improved the electrochemically active surface area of carbon paper, which was favorable for the adhesion of electrochemical active bacteria. MFC-Fe@CN achieved a maximum voltage output of 792.76 mV, which was 28.61% higher than that of MFC-CP (616.39 mV). Moreover, MFC-Fe@CN presented a longer stable voltage output, while that of MFC-CP decreased to about 0 mV in each cycle. Such extraordinary performance of MFC-Fe@CN was ascribed to three major aspects: (a) the Fe@CN modified anode enhanced both of the electrochemically active surface area (EASA) and electrochemical activity of EABs for more active interfacial interaction between exoelectrogens and electrode. (b) the higher content of biomass (7.27 mg/cm²) and EPS (2.69 mg/cm²) with a higher proteins/polysaccharides ratio in MFC-Fe@CN finely regulated by cells to keep a balance between EET efficiency and self-protection against environmental stress. (c) Geobacter species, the well-known exoelectrogens, was selectively enriched in abundance in MFC-Fe@CN, which was particularly beneficial for efficient electrons generation. The present study provided a new insight into the use of Fe@CN nanoparticles for enhancing the bioelectrochemical performance of MFCs, which may contribute to the practical application of MFCs in energy generation and wastewater treatment.

Acknowledgements

Funding

This work was supported by the National Key Research and Development Program of China (Project No. 2021YFA0910400), the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (Project No. XTD2216), the Graduate Research and Innovation Projects of Jiangsu Province (Project No. KYCX22_1349), the Jiangsu Province Science and Technology Program Special Fund (Hong Kong, Macao and Taiwan Science and Technology Cooperation) (BZ2022052), and the National Natural Science Foundation of China (Project No. 21777069).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Xiao-Long Cheng: Conceptualization, Methodology, Visualization, Writing - original draft. Qiang Xu: Data curation. Qian-Wen Yang: Investigation. Rui-Rui Tian: Investigation. Biao Li: Data curation Investigation, characterization. Su Yan: Project administration. Xue-Ying Zhang: Project administration. Jun Zhou: Project administration. Xiao-Yu Yong: supervision, resources, funding acquisition, writing - review & editing.

Ethical Approval

Not applicable

Consent to Participate

Not applicable

Consent to Publish

Not applicable

Availability of data and materials

The datasets generated or analyzed during this study are available from the corresponding author on reasonable request.

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Scheme 1 is available in the Supplementary Files section.

Scheme1.png
Scheme 1. Pyrolytic synthesis of Fe@CN nanospheres.
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Editorial decision: Major Revision
27 Oct, 2022
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You are reading this latest preprint version

Enhancing extracellular electron transfer through selective enrichment of Geobacter with Fe@CN modified carbon-based anode in microbial fuel cells

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Abstract

Figures

Introduction

Materials And Methods

One-pot method for the synthesis of Fe@CN nanospheres

Characterization Of Catalyst

Electrode Fabrication And Mfcs Setup

Characterization And Analytical Measurements

Results And Discussion

Characterization of Fe@CN catalysts

Electrochemical Active Surface Area Of Different Anodes

Bioelectrochemical Performances Of Mfcs With Fe@cn Modified Anode

The Structure And Morphology Of Eabs On The Anodes Modified With Fe@cn

The Modified Anodes Modulated The Eps Production Of Eabs

Microbial Community Analysis Of Eabs On Fe@cn Modified Anodes

Conclusions

Declarations

References

Scheme 1

Supplementary Files

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Version 1