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Article

Porous Carbon Derived from Pumpkin Tissue as an Efficient Bioanode Toward Wastewater Treatment in Microbial Fuel Cells

by
Jiaxin Liu
1,2,
Xue Yan
3,
Qiang Ding
4,
Jiwu Xiang
4,
Zuna Wei
1,2,
Qian Yang
1,2,
Kangwei Xie
1,2,
Bo Cheng
1,2,* and
Xiaoying Xie
5,*
1
School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China
2
Hubei Key Laboratory of Microbial Transformation and Regulation of Biogenic Elements in the Middle Reaches of the Yangtze River, Wuhan 430205, China
3
Yichang Humanwell Pharmaceutical Co., Ltd., Yichang 443005, China
4
Yichang Sanxia Pharmaceutical Co., Ltd., Yichang 443004, China
5
Tianjin Key Laboratory of Structure and Performance for Functional Molecules, College of Chemistry, Tianjin Normal University, Tianjin 300387, China
*
Authors to whom correspondence should be addressed.
Sustainability 2025, 17(11), 4758; https://doi.org/10.3390/su17114758
Submission received: 13 April 2025 / Revised: 25 April 2025 / Accepted: 14 May 2025 / Published: 22 May 2025
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Abstract

:
A novel three-dimensional porous biocarbon electrode with exceptional biocompatibility was synthesized via a facile approach using pumpkin as the precursor. The obtained pumpkin-derived biocarbon features a highly porous architecture and serves as an efficient biocarbon electrode (denoted as PBE) in a microbial fuel cell (MFC). This PBE could form robust biofilms to facilitate the adhesion of electroactive bacteria. When used in the treatment of real wastewater, the assembled PBE-MFC achieves a remarkable power density of 231 mW/m2, much higher than the control (carbon brush—MFC, 164 mW/m2) under the identical conditions. This result may be attributed to the upregulation of flagellar assembly pathways and bacterial secretion systems in the electroactive bacteria (e.g., Hydrogenophaga, Desulfovibrio, Thiobacillus, Rhodanobacter) at the anode of the PBE-MFC. The increased abundance of nitrifying bacteria (e.g., Hyphomicrobium, Sulfurimonas, Aequorivita) and organic matter-degrading bacteria (e.g., Lysobacter) in the PBE-MFC also contributed to its exceptional wastewater treatment efficiency. With its outstanding biocompatibility, cost-effectiveness, environmental sustainability, and ease of fabrication, the PBE-MFC displays great potential for application in the field of high-performance and economic wastewater treatment.

Graphical Abstract

1. Introduction

Currently, conventional activated sludge, with energy-intensive operation for treating urban domestic wastewater, emits lots of greenhouse gases, such as CO2, CH4, and N2O [1,2]. In addition, the process generates substantial volumes of wastewater effluent, leading to considerable economic burdens. Microbial fuel cells (MFCs), a novel wastewater treatment technology, utilize electroactive microorganisms as biocatalysts to convert the chemical energy present in wastewater into electrical energy. This innovative approach addresses the limitations of traditional treatment methods while aligning with the principles of waste-to-resource recycling [3]. With the advantages of the broad availability of raw materials, high resource efficiency, simplicity of operation, low environmental impact, and simultaneous wastewater treatment and power generation, MFCs have garnered extensive attention for their potential in pollutant removal and recovery across diverse wastewater streams [4].
However, the power density of MFCs remains constrained by multiple interdependent factors, including electrode material properties, biofilm formation dynamics, microbial community composition, system architecture, and operational parameters [5]. The scalable deployment of MFCs continues to face critical challenges, primarily stemming from factors including irreversible membrane fouling, which degrades ion transport efficiency, cost-intensive reliance on noble metal cathodic catalysts (e.g., platinum), and operational instability during prolonged current cycling. A persistent challenge stems from the inherent limitations of conventional anode materials—specifically, their excessive production costs and non-renewable nature—which collectively restrict electron transfer efficiency and thereby limit practical scalability. This bottleneck directly compromises power generation capacity while simultaneously hindering the technology’s transition from laboratory prototypes to real-world applications. Electrode material costs are estimated to constitute approximately 20–50% of the total expenses associated with MFCs, highlighting the need for economical alternatives [6]. Consequently, the development of high-performance anode materials has garnered significant research attention. In recent years, there has been a great deal of research on the different anode materials used for MFCs [7]. Traditional carbon-based materials, such as carbon felt, carbon brush (CB), carbon cloth, carbon paper, and graphite, have been widely used as anode materials in MFCs due to their high specific surface area, excellent conductivity, and chemical stability. However, these materials face several limitations, including poor hydrophilicity, two-dimensional structures, and low catalytic activity [8]. To address these challenges, modifying these carbon-based materials is a common strategy to enhance their electrical performance, but it suffers from high costs, which restricts their scalability and widespread adoption.
Porous carbon materials have received increasing attention due to their large reaction surface area, short diffusion paths, and reduced diffusion effects [9]. At present, the preparation of porous carbon electrodes using various biomass materials as carbon sources [10] has become a research hotspot in the field of energy storage and conversion, including cotton pulp, lignin, silk, and peanut shells [11,12]. These biomass-derived materials, characterized by a three-dimensional (3D) porous structure, facilitate internal colonization and enhance bacterial adhesion. Moreover, biomass-based carbon electrodes are typically pyrolyzed at high temperatures (700–900 °C), resulting in the formation of hydroxyl and carboxyl functional groups on their surfaces [13]. These functional groups improve surface hydrophilicity, promoting the formation of electrochemically active biofilms [14]. So, biomass-derived porous carbon electrode materials not only enhance electrochemical performance but also significantly reduce costs and toxicity [15].
Biomaterials offer significant advantages as they are renewable, environmentally friendly, cost-effective, and highly versatile, making them promising candidates for MFC applications. For instance, Yong Yuan et al. [16] developed an open three-dimensional electrode using natural loofah sponge as a precursor material, achieving a power density of 1090 ± 72 mW/m2, surpassing that of traditional three-dimensional electrodes. The bioanode developed by Zhao et al. presented a novel strategy for enhancing MFC performance [17]. Owing to the unique properties of bio-magnetic carbonized loofah cellulose fibers, the bioanode demonstrated remarkable electrical output and energy storage capabilities. This innovation also offers a promising solution to the voltage reversal issue in stacked MFCs and is expected to advance the development of next-generation bioenergy technologies. Similarly, Yini Chen et al. fabricated bioanodes from wax gourd, which demonstrated a higher maximum power density compared to a control carbon felt anode in MFCs [18]. A highly biocompatible three-dimensional hierarchically porous activated carbon, derived from Miscanthus sacchariflorus, exhibited an unprecedented specific surface area of 3027 m2/g when employed in an MFC [19]. Clearly, the research on biomass-derived carbon materials provides new avenues for the high-value-added utilization of biomass resources. This, in turn, ultimately helps in the rational design of more efficient biomass-based carbon materials for application in MFCs.
However, the explored biomass materials were severely restricted in terms of further industrialization, mainly due to their limited, costly raw materials, geographical distribution, and difficulties associated with their cultivation, transportation, and storage. In contrast to most other precursors, pumpkins are rich in sugars and starches and possess favorable characteristics for large-scale production worldwide. As a renewable and low-cost precursor, pumpkins were first utilized by Suying Bai et al. to prepare porous carbon materials with exceptional properties [20]. The resulting pumpkin-derived activated carbon materials exhibited a high specific surface area of 2968 m2/g, along with abundant micropores and mesopores. Moreover, they also showed remarkable cycling stability. Consequently, pumpkin-derived carbon materials, which are easily accessible and possess superior electrochemical properties, emerge as promising electrode candidates for MFCs.
Herein, a novel, highly biocompatible 3D pumpkin-derived biocarbon porous electrode (denoted as PBE) was synthesized using a simple method. The surface morphology and material properties of the prepared PBE were thoroughly investigated. Subsequently, the power generation and wastewater treatment capabilities of the PBE were evaluated in an MFC inoculated with real wastewater and compared with those of a commercial CB electrode. This study reports the inaugural successful application of the proposed PBE in an MFC for wastewater treatment. It positions pumpkin as a low-cost, high-performance alternative to traditional carbon electrodes, promoting the design of MFCs for wastewater treatment.

2. Materials and Methods

2.1. Electrode Preparation

The preparation process of the PBE was improved in previous studies demonstrating optimal pore formation and graphitization for biomass-derived electrodes [13,20]. Fresh pumpkins were purchased from a local market in Wuhan, Hubei Province, China. The preparation of the PBE involved the following steps: first, the pumpkin was cut into cylindrical pieces with a diameter of 3 cm and a thickness of 4 cm. The sample was then placed in an ultralow-temperature freezer and frozen at −80 °C for 48 h. Subsequently, the frozen pumpkin sample was subjected to freeze-drying using a vacuum dryer (SCIENTZ-18N, NingBo Scientz, Ningbo, China) at −50 °C for 72 h. Then, the freeze-dried pumpkin was subjected to carbonization in a N2 atmosphere. The material was compressed and heated in a tubular furnace at 800 °C for 3 h, after which the carbonized electrode was connected to titanium wire for application in the MFC. For comparison, the control anode (CB; dimensions: Φ 2 × 6 cm) was soaked in a 1 M H2SO4 solution for 24 h, thoroughly washed with deionized water until neutral, and dried at 60 °C for 24 h.

2.2. Characterization

The morphology of the prepared electrodes, including the PBE and CB, was characterized using scanning electron microscopy (SEM, Gemini SEM 300, Zeiss, Obekochen, Germany). The morphological and structural characteristics of the PBE were systematically investigated utilizing transmission electron microscopy (TEM, JEM-2100 model, JEOL Ltd., Tokyo, Japan). To precisely determine the specific surface area (SSA) and pore size distribution of the PBE, we employed an automated SSA and micropore analyzer (ASAP 2020 HD88, Micromeritics, Norcross, GA, USA). SSA calculations were performed in combination with the Brunauer–Emmett–Teller (BET) theory, and pore size analysis was performed using the Barrett–Joyner–Halenda (BJH) method. The measurements were conducted under degassed conditions (200 °C for 8 h) to eliminate adsorbed contaminants, thereby guaranteeing the accuracy and reproducibility of the data. The crystal structure was evaluated via X-ray diffraction (XRD, D8 ADVANCE, Bruker, Berlin, Germany). The elemental composition was determined using X-ray photoelectron spectra (XPS, ESCALAB XI+, Thermo Fisher Scientific, Carlsbad, CA, USA). Subsequently, the functional groups present in the materials were examined through a Fourier transform infrared spectrometer (FTIR, Nicolet 6700, Thermo Fisher Scientific, Carlsbad, CA, USA) across a scanning range of 4000~400 cm−1. Additionally, further structural analysis of the PBE was performed employing a laser confocal micro-Raman spectrometer (DXR model, Thermo Fisher Scientific, Carlsbad, CA, USA).

2.3. MFC Setup and Operation

In this study, a dual chamber (a volume of 650 mL for both anode and cathode) was constructed with the PBE and CB as the anode and cathode, respectively, and separated by a proton exchange membrane. The anode and cathode were spaced 12 cm apart, connected by a copper wire, and operated with an external resistance of 9999 Ω. For comparison, the CB served as an anode in the control MFC with the same configuration as the experimental group. The anode chambers of two MFCs were inoculated with sludge from the biochemical tank of a domestic sewage treatment plant in Zhongxiang, serving as the microbial catalyst. The inoculum was mixed with domestic wastewater in a 1:3 ratio to prepare the anode solution. The cathode chamber contained a 50 mM potassium ferricyanide–phosphate buffer solution (PBS, pH = 7.2) as the electrolyte. To maintain aerobic conditions, the anode electrolyte was purged with sterile air and replaced every 48 h. The MFCs were operated in fed-batch mode at 25 °C, and the cell voltages were recorded through the electrochemical workstation (Chi760e, CH Instruments, Shanghai, China) every 10 s. When the maximum voltages were reproducible over three consecutive cycles, the MFC start-ups were considered successful.

2.4. Pollutant Removal

According to the Chinese National Standard (HJ535-2009) [21], Nessler’s reagent spectrophotometry was used to detect the ammonia nitrogen (NH4+-N) content in domestic sewage. Following the Chinese National Standard (GB11893-1989) [22], ammonium molybdate spectrophotometry was used to determine the total phosphorus (TP) content in sewage. Based on the Chinese National Standard (HJ 924-2017) [23], potassium dichromate rapid digestion photometry was used to determine the chemical oxygen demand (COD) in wastewater. All the above pollutant indices were determined with a portable water quality rapid detector (T3WS, Beijing Purkinje, Beijing, China), ensuring consistency and accuracy across measurements. All experiments were conducted in triplicate, with quantitative data expressed as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA, USA) under a rigorous analytical framework. Intergroup comparisons were analyzed through one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) post hoc test. Statistical significance was denoted as follows: ns (not significant, p > 0.05), * (0.01 < p ≤ 0.05), ** (0.001 < p ≤ 0.01), *** (0.0001 < p ≤ 0.001), and **** (p ≤ 0.0001).

2.5. Microbial Community Analysis

Microbial samples were collected on the final day of MFC operation. A portion of the biofilm on the PBE was carefully scraped off under ultra-clean conditions using sterilized scissors. In the case of the CB, sterilized scissors were used to trim sections of the bristles with attached biofilm. The obtained biofilms were divided into two subsets for analysis. One subset was immediately flash-frozen in liquid nitrogen for 30 min and stored at −80 °C for subsequent 16 S rDNA sequencing, which was conducted by Shenzhen Chengqi Biotechnology Co., Ltd. ( Shenzhen, China). The other subset underwent fixation in a 2.5% glutaraldehyde solution for 4 h, followed by three washes with PBS buffer (0.1 mol/L, pH 7.2). The samples were then subjected to a graded ethanol dehydration series (30%, 50%, 75%, and 100%) for 20 min at each concentration. Finally, the samples were dried at 60 °C in an oven and analyzed via SEM to characterize microbial morphology at the microstructural level.

3. Results and Discussion

3.1. Characterization of the PBE

The resulting black PBE retains the macroscopic characteristics of dried pumpkins with a volume shrinkage of ~20% after carbonization. The SEM images in Figure 1a–c show that the fractured PBE possesses an interconnected network with irregular pore structure, which may result from the decomposition of organic matter and the volatilization of water during the carbonization process. Following high-temperature carbonization, the surface structure of the material is rougher compared to the planar structure of carbon-based materials. The TEM images (Figure 1d–f) reveal that the PBE has a nanoporous structure. Figure 2 also reveals that the PBE exhibits a hierarchical pore architecture comprising macropores (>50 nm), mesopores (2–50 nm), and micropores (<2 nm), with a BET SSA of 337.66 m2/g. This multimodal porosity enables synergistic optimization of biotic and abiotic processes: (i) micropores facilitate bacterial quorum sensing through confined molecular signaling, (ii) mesopores enhance biofilm matrix permeation via capillary-driven hydration, and (iii) macropores permit direct microbial colonization. Compared to planar carbon substrates, this 3D porous framework significantly amplifies the electroactive surface area, providing abundant microbial anchoring sites while improving mass transfer kinetics critical for sustained electrocatalytic activity. These properties are expected to contribute to the increased power density observed in MFCs. Notably, the solvent-free fabrication process eliminates the energy-intensive chemical activation steps typically required for biomass-derived electrodes.
The XRD pattern for the PBE in Figure 3a shows two broad peaks of graphitic carbon around 26° and 40°. The XRD peak of graphite carbon, observed around 26°, is commonly attributed to the (002) crystal plane of graphite. The diffraction peak at approximately 40° typically corresponds to other atypical diffraction features within graphite, which may be related to the (101) or (110) crystal planes of graphite. These results confirm that the pumpkin biomass was successfully transformed into a carbon material during the carbonization process. The XPS spectrum in Figure 3b reveals the existence of C, N, and O in the PBE. As depicted in the FTIR spectrum (Figure 3c), the O–H stretching vibration at 3459 cm−1, along with stretching vibrations of C = O (1634 cm−1) and C–O (1030 cm−1), demonstrate the presence of hydroxyl and carboxyl groups on the PBE surface. Raman spectroscopy reveals characteristic peaks in the sample at approximately 1350 cm−1 and 1580 cm−1 (Figure 3d), attributed to the D (defect and disorder) and G (graphite) bands of carbon materials. The ID/Ig ratio of 1.02 indicates a high degree of graphitization in this carbon material, yet it still contains certain defects and disordered regions, such as sp3 hybridization, edge defects, or grain boundaries. They all enhance its hydrophilicity and promote the formation of electrochemically active biofilms [10]. Oxygen-containing functional groups on the surface of biocarbon materials, such as carboxyl and hydroxyl groups, can interact with proteins or polysaccharides on microbial cell surfaces through hydrogen bonds [24]. These interactions enhance the adhesion of microbial cells to the biocarbon material surface, thereby strengthening the adhesion and stability of the biofilm [25]. Prior research has demonstrated that introducing oxygen-containing functional groups on carbon-based bioanodes can boost cytochrome c expression and accelerate the extracellular electron transfer rate [26]. Cytochrome c, a key protein in microbial extracellular electron transfer, plays a crucial role in this process [27]. These combined performance metrics and sustainable manufacturing characteristics position the PBE as an alternative material for next-generation bioelectrochemical systems in wastewater remediation and energy harvesting applications.

3.2. Bioelectricity Generation in MFCs

After 7 days of operating the MFCs, polarization measurements were conducted at the peak of the open-circuit potential to evaluate electrochemical performance. The electrochemical workstation was used to analyze the electrochemical performance of the PBE-MFC and CB-MFC. The analysis employed a range of external resistors (1 kΩ to 100 Ω), and the cell voltage across each resistor was monitored during the peak voltage stabilization phase. The power density and polarization curves of the MFCs are plotted by voltammetry, as illustrated in Figure 4a,b.
As shown in Figure 4a, when the external resistance decreases from 9000 Ω to 500 Ω, the power density of the PBE-MFC increases rapidly from 31 to 231 mW/m2, accompanied by a corresponding output voltage of 370 mV. However, as the external resistance further decreases to 100 Ω, the power density gradually declines to 121 mW/m2. The power density peaks when the external and internal resistances are equal and decreases when internal polarization is achieved. Similarly, the power density of the CB-MFC follows a similar trend, peaking at an external resistance of 500 Ω (Figure 4b). The CB-MFC achieves a lower maximum power density of 131 mW/m2 with a corresponding output voltage of 281 mV, highlighting the superior performance of the PBE-MFC under identical conditions. Compared to other reported biomass-derived carbon anodes [19], both MFCs suffer lower power densities due to the use of actual wastewater in this study without any addition of extra carbon sources or nutrients. The inherent complexity of substances in real wastewater can inhibit both the growth of electroactive bacteria and the electron transfer processes. Nevertheless, under identical wastewater and experimental conditions, the higher peak power density generated by the PBE-MFC in real wastewater (231 mW/m2) than that of the CB-MFC (164 mW/m2) underscores the advantages of the PBE as an anode material over the CB in MFC-based wastewater treatment. It should be noted that additional electrochemical analyses could provide further insights into the charge transfer and capacitive behavior of the PBE compared to the CB, which will be considered in future work.

3.3. Performance of the PBE-MFC

Under a hydraulic retention time of 48 h, the PBE-MFC achieved a COD removal rate of 71.43%, significantly outperforming the CB-MFC with a removal rate of 58.79% (Table 1). The metabolic activity of potential organic-matter-degrading microorganisms within the anodic biofilm facilitates the breakdown of organic pollutants in wastewater. The degradation of COD occurs in two primary stages: the catabolic degradation (oxidative breakdown) of organic matter and the anabolic synthesis (growth and proliferation) of microorganisms. Under aerobic conditions, aerobic microorganisms utilize dissolved oxygen as the electron acceptor to oxidize and decompose biodegradable organic compounds in the wastewater. As both carbon and energy sources, organic compounds are enzymatically oxidized by microorganisms into simple, low-molecular-weight compounds, with carbon dioxide and water as the final byproducts. A portion of the organic matter serves as raw material for microbial anabolic processes, ultimately being converted into new cellular biomass. COD is a critical indicator for assessing the level of organic pollution in water. A high COD removal rate signifies that the microorganisms on the PBE-MFC anode are capable of efficiently degrading organic pollutants in wastewater into harmless substances. This further demonstrates the strong wastewater treatment capacity of the PBE-MFC, which contributes to enhancing the usability of water and mitigating the environmental impact of organic pollutants.
For NH4+-N removal (Table 1), the PBE-MFC achieved a remarkable efficiency of 70.18%, surpassing the 53.75% observed in the CB-MFC. It is known that NH4+-N is a common pollutant in wastewater, and excessive concentrations can be detrimental to aquatic ecosystems. The PBE-MFC demonstrates a high removal efficiency, indicating its capability to effectively reduce NH4+-N through electrochemical reactions and microbial metabolic processes. This not only mitigates the toxicity of the water but also contributes to the overall improvement in water quality. This enhanced performance could be attributed to the activity of nitrifying bacteria, which facilitate the oxidation of NH4+-N into NO3− through two sequential steps. In the first step, ammonia-oxidizing bacteria oxidize NH4+-N to NO2− via the reaction: NH4+ + 1.5 O2 → NO2− + 2H+ + H2O. Subsequently, nitrite-oxidizing bacteria further oxidize nitrite NO2− to NO3− according to the reaction: NO2− + 0.5 O2 → NO3−. Similarly, the PBE-MFC demonstrated an enhanced TP degradation efficiency in domestic sewage, with a degradation rate of 67.40%, higher than 53.30% for the CB-MFC (Table 1). It is well-known that under aerobic conditions, certain polyphosphate-accumulating organisms within a biofilm are capable of assimilating phosphate from wastewater, thereby contributing to the reduction in TP content. Furthermore, this plays a crucial role in mitigating the issue of eutrophication in aquatic systems, thereby contributing to the protection of water ecosystems.
In summary, under comparable operational conditions and identical wastewater retention parameters, the PBE-MFC consistently demonstrated superior overall wastewater treatment performance. This enhanced efficiency can be attributed to the intrinsically porous structure of the PBE, coupled with the presence of hydroxyl and carboxyl groups on its surface, which confer improved hydrophilicity and biocompatibility. On the one hand, oxygen-containing functional groups can act as active sites to adsorb organic matter and nutrients in wastewater, thereby enhancing the degradation efficiency of pollutants by the biofilm. Furthermore, these groups can influence the types and distribution of microorganisms within the biofilm, optimizing the microbial community structure and improving the treatment capacity of the biofilm [28]. Research has demonstrated that an increase in the surface oxygen content is crucial for promoting the enrichment of nitrifying bacteria on carbon-based cathode surfaces in biofilm electrode reactors [29]. Compared to the CB, these properties facilitate the preferential enrichment of certain functional bacteria on the PBE. Such bacterial colonization significantly enhances critical processes, including organic matter degradation and nitrification, thereby driving the superior wastewater treatment efficiency of the PBE-MFC. These findings underscore the potential of the PBE-MFC as a more effective and sustainable solution for advanced wastewater treatment applications.

3.4. Biofilm on Anode

The SEM images (Figure 5a–c) of the electroactive biofilms on the PBE show the prolific colonization of electroactive bacteria within the electrode’s pores and crevices. These bacteria develop robust biofilms with densely packed and interconnected spherical structures. The spherical bacteria exhibit uniform morphology, smooth surfaces, and high density, indicative of significant aggregation. The electroactive bacteria form intimate contact with the electrode surface, thereby reducing the impedance of electron transfer and enhancing the electrode’s conductivity. In summary, the PBE anode exhibits an increased electrode contact area and a stable biofilm structure. These features significantly improved the system’s power generation, underscoring their critical role in the performance of the PBE-MFC.
To further elucidate the mechanisms behind the superior power density and enhanced wastewater treatment efficiency of the PBE-MFC, 16 S rDNA sequencing was conducted to analyze the microbial communities. Universal primers targeting conserved regions of the 16 S rDNA were employed for PCR amplification, followed by paired-end sequencing of one or more hypervariable regions using the Illumina MiSeq/HiSeq platforms. The resulting sequences from hypervariable regions were clustered into Operational Taxonomic Units (OTUs) based on a 97% similarity threshold using VSEARCH software 2.3.4, enabling the definition of OTUs. Representative sequences for each OTU were taxonomically annotated to the species level with the RDP Classifier (Version 2.2) against the Greengenes database. Furthermore, comprehensive analyses of species composition and relative abundance were performed across different taxonomic hierarchies to elucidate the microbial diversity and community structure in each sample.
Linear Discriminant Analysis (LDA) was employed through LEfSe software to identify taxa with significantly different abundances between experimental groups [30]. Unlike Principal Component Analysis (PCA), which provides an unsupervised projection of the dataset onto axes that maximize overall variance without accounting for classification information, LDA is a supervised approach. By integrating inter-taxa relationships with feature selection, LDA combines standard significance tests (Kruskal–Wallis and pairwise Wilcoxon tests) with linear discriminant analysis to identify and rank features of biological importance. This method not only pinpoints taxa critical to group differentiation but also ranks their functional contributions based on effect size, offering a more comprehensive explanation of observed biological disparities. In this study, a p-value threshold of 0.05 and an LDA score above 2 were set as criteria for significance. The results, presented in Figure 6, highlight taxa with significant differential abundances at the genus level.
Figure 6 reveals an increased abundance of several functional bacterial genera within the PBE-MFC anode, including Lysobacter, Hydrogenophaga, Desulfovibrio, Thiobacillus, Rhodanobacter, Hyphomicrobium, Methylocaldum, Azospirillum, Sulfurimonas, Aequorivita, etc. Lysobacter is a decarbonizing microorganism capable of breaking down organic matter in water [31]. Notably, Desulfovibrio, a heterotrophic exoelectrogen, preferentially adheres to the fixed surfaces of biochar anodes [32,33], whereas Hydrogenophaga, an autotrophic electro-producing bacterium, exists in both fixed and suspended forms but is selectively enriched on the biochar surface. This suggests the potential establishment of a syntrophic relationship between Desulfovibrio and Hydrogenophaga on the PBE surface, facilitating efficient energy harvesting [34]. Both Rhodanobacter [35,36] and Thiobacillus [37], previously identified as electroactive genera in MFCs, play pivotal roles in electron transfer. Additionally, Thiobacillus also functions as an electroactive denitrifying microorganism, capable of utilizing electrons as electron donors and carbon dioxide as a carbon source during denitrification. Previous studies have identified Sulfurimonas and Thiobacillus as key sulfur-utilizing denitrifying bacteria [38]. Hyphomicrobium performs both heterotrophic nitrification and aerobic denitrification, achieving simultaneous nitrification and denitrification through ammonia oxidation coupled with nitrate reduction [31]. Methylocaldum, a member of the Pseudomonadota phylum, constitutes a significant portion of the electrogenic bacterial community on the anode [39]. While the mechanisms of indirect electron transfer by Methylocaldum remain uncertain, some strains reportedly harbor outer membrane cytochrome (OMC) genes associated with electron transfer to Fe (III) oxides [40]. Extracellular electron transfer to Fe (III) oxides is closely linked to the function of OMCs in electroactive bacteria. Genes encoding cytochrome-like proteins are predominantly expressed in species such as Methylocystis, Methylocaldum, and Methylobacter, indicating their involvement as electron donors in interspecies electron transfer. This mechanism may involve an OMC-based conductive matrix. Azospirillum, a nitrogen-fixing bacterium, is an uncommon member of electroactive consortia [41]. Nevertheless, some studies [42] report that Azospirillum strains isolated from electrodes can perform extracellular respiration of anthraquinone-2,6-disulfonate (AQDS), a typical redox mediator for electroactive bacteria [43]. Finally, Aequorivita demonstrates denitrification capabilities [37], possessing the nos gene required to convert N2O to N2, thereby mitigating N2O emissions [44].
Tax4Fun is an advanced R package developed for the functional prediction of environmental samples, including those from intestinal and soil ecosystems, leveraging the 16 S SILVA database [45]. Demonstrating superior predictive accuracy compared to PICRUSt, Tax4Fun has proven particularly effective for analyzing complex environments, such as soil. The functional prediction capability of Tax4Fun is achieved through a nearest-neighbor approach based on minimal 16 S rRNA sequence similarity. Specifically, the method involves extracting the 16 S rRNA gene sequences from prokaryotic genomes in the KEGG database and aligning them to the SILVA SSU Ref NR database using the basic local alignment search tool (BLAST) algorithm (with a BLAST bitscore threshold > 1500) to construct a correlation matrix. Functional information annotated within the KEGG database—using the UProC and PAUDA tools—is subsequently mapped onto the SILVA framework, enabling comprehensive functional annotation of the SILVA database. OTUs clustered from the sequencing samples were annotated against the SILVA database to obtain corresponding functional annotations. Based on these database annotations, differential analysis between groups was conducted using t-tests, selecting pathways with p-values below 0.05 for visualization in Figure 7. The left panel of Figure 7 highlights the differential functional abundance between groups, where each bar represents the mean abundance of functions with significant differences across groups. The right panel displays the confidence intervals of these differences, with each circle’s leftmost point indicating the lower bound and the rightmost point the upper bound of the 95% confidence interval for the mean difference. Furthermore, the circle’s color denotes the group exhibiting higher functional abundance. The far right section of the figure reports p-values derived from statistical significance tests of differential taxa between groups.
Our finding indicates a significant enrichment of the flagellar assembly pathway in the PBE-MFC (adjusted p-value = 0.00015). Pili are extremely fine extracellular filaments located on the bacterial cell wall, serving as critical appendages for bacterial motility, surface colonization, and electrode respiration. In electroactive bacteria, conductive pili play an essential role in the attachment of electrons and their subsequent transfer to electrodes [46]. Reguera and colleagues reported the electrical conductivity of Type IV pili in Geobacter sulfurreducens, referring to them as nanowires [2]. Despite the fact that the conductive pili of Shewanella oneidensis MR-1 are only a few micrometers in length, they exhibit an exceptional electron transfer rate of 109/s [47].
Additionally, the bacterial secretion system was markedly upregulated in the PBE-MFC (adjusted p-value = 0.00005). These systems are intricate molecular machinery that facilitate the transport of various substances, including small molecules and proteins, across the bacterial cell envelope. Biofilms are enriched with polysaccharides, proteins, nucleotides, and other cellular secretions, collectively known as the extracellular polymeric substance (EPS) matrix. EPS is a fundamental microbial component that governs the physicochemical properties of biofilms. Nearly every microbial cell is encased in EPS, which aids in biofilm formation and provides protection against adverse environmental conditions. In electroactive microorganisms, the EPS matrix facilitates direct bacteria–electrode interactions and establishes robust connections between bacterial cells and the anode surface [48]. Previous studies have demonstrated that redox-active molecules within the EPS core facilitate extracellular electron transfer, and EPS can serve as an electron conduit for interspecies electron transfer among electroactive bacteria [49]. Notably, bacterial secretion systems are closely intertwined with EPS, playing indispensable roles in the synthesis, transport, assembly, and regulation of this matrix. These interactions are fundamental to the formation and functionality of electroactive biofilms. Accordingly, the marked upregulation of the bacterial secretion system in the PBE-MFC is likely a key driver of enhanced bioelectrochemical activity in electroactive bacteria, ultimately contributing to the increased power density observed in the system.

4. Conclusions

Herein, a novel 3D hierarchical porous biocarbon electrode with high biocompatibility was synthesized using pumpkin as a precursor through a simple and efficient method. In MFCs inoculated with real wastewater, the PBE demonstrated superior power generation and wastewater treatment performance compared to the CB anode. Relative to the CB-MFC, the PBE-MFC exhibited an upregulation in the abundance of functional bacterial genera, including electroactive bacteria (e.g., Hydrogenophaga and Desulfovibrio), nitrifying bacteria (e.g., Hyphomicrobium and Sulfurimonas), and organic-matter-degrading bacteria (e.g., Lysobacter). Tax4Fun functional group analysis of the microbiota revealed that the power density of the PBE-MFC was 1.4 times higher than that of the control group. The significant upregulation of flagellar assembly pathways in electroactive bacteria, along with enhanced bacterial secretion systems, may have contributed to accelerated electron transfer. This study offers new insights into the design of hydrophilic, biocompatible, and cost-effective MFC anodes using natural biomass. Next-generation material engineering strategies leveraging nanomaterial-enabled surface modification protocols demonstrate potential to synergistically enhance both SSA and electrochemical cycling stability. Crucially, systematic investigation of the long-term operational robustness of PBE-MFC substrates—particularly their biofilm durability under fluctuating organic loads—is imperative to address scalability barriers in high-capacity wastewater treatment applications.

Author Contributions

J.L.: Conceptualization, data curation, formal analysis, investigation, methodology, software, writing—original draft, and writing—review and editing. X.Y.: data curation, formal analysis, investigation, software, and writing—review and editing. Q.D.: formal analysis, investigation, and writing—review and editing. J.X.: formal analysis, investigation, and writing—review and editing. Z.W.: data curation, formal analysis, investigation, software, and writing—review and editing. Q.Y.: data curation, software, and writing—review and editing. K.X.: formal analysis, writing—review and editing. B.C.: conceptualization, methodology, visualization, writing—review and editing. X.X.: methodology, visualization, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Yichang Sanxia Pharmaceutical Co., Ltd. [grant number 2023BBB120] and the National Natural Science Foundation of China [grant number 22209121].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

X.Y. was employed by Yichang Humanwell Pharmaceutical Co., Ltd. Q.D. and J.X. were employed by Yichang Sanxia Pharmaceutical Co., Ltd. The remaining authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. SEM images of the PBE: (a) 40 μm, (b) 20 μm, and (c) 10 μm. TEM images of the PBE: (d) 50 nm, (e) 20 nm, and (f) 10 nm.
Figure 1. SEM images of the PBE: (a) 40 μm, (b) 20 μm, and (c) 10 μm. TEM images of the PBE: (d) 50 nm, (e) 20 nm, and (f) 10 nm.
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Figure 2. The nitrogen adsorption/desorption isotherms and pore size distributions of the PBE.
Figure 2. The nitrogen adsorption/desorption isotherms and pore size distributions of the PBE.
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Figure 3. (a) XRD spectra, (b) XPS spectra, (c) FTIR spectra, and (d) Raman spectra of the PBE.
Figure 3. (a) XRD spectra, (b) XPS spectra, (c) FTIR spectra, and (d) Raman spectra of the PBE.
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Figure 4. The polarization curves and power density of the PBE-MFC (a) and CB-MFC (b).
Figure 4. The polarization curves and power density of the PBE-MFC (a) and CB-MFC (b).
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Figure 5. SEM images of the PBE in MFC: (a) 10 μm, (b) 2 μm, and (c) 1 μm.
Figure 5. SEM images of the PBE in MFC: (a) 10 μm, (b) 2 μm, and (c) 1 μm.
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Figure 6. Heatmap of differential contribution analysis based on LDA.
Figure 6. Heatmap of differential contribution analysis based on LDA.
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Figure 7. Differential functional group analysis at level 3 using Tax4Fun.
Figure 7. Differential functional group analysis at level 3 using Tax4Fun.
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Table 1. Pollutant removal performance of the PBE-MFC and CB-MFC. Significance levels: ns (p > 0.05), * (0.01 < p ≤ 0.05), ** (0.001 < p ≤ 0.01), *** (0.0001 < p ≤ 0.001), and **** (p ≤ 0.0001).
Table 1. Pollutant removal performance of the PBE-MFC and CB-MFC. Significance levels: ns (p > 0.05), * (0.01 < p ≤ 0.05), ** (0.001 < p ≤ 0.01), *** (0.0001 < p ≤ 0.001), and **** (p ≤ 0.0001).
PollutantTimeCB-MFCPBE-MFCSignificance Between CB-MFC and PBE-MFC
COD
(mg/L)		0 h182.33 ± 7.51180.00 ± 12.29ns
(Adjusted p = 0.9808)
48 h75.00 ± 3.0052.00 ± 4.00*
(Adjusted p = 0.0252)
Significance Before/After Treatment****
(Adjusted p < 0.0001)		****
(Adjusted p < 0.0001)		-
Average Removal Rate58.79%71.43%****
(Adjusted p < 0.0001)
NH4+-N
(mg/L)		0 h4.84 ± 0.184.92 ± 0.28ns
(Adjusted p = 0.9502)
48 h2.28 ± 0.131.43 ± 0.19**
(Adjusted p = 0.0040)
Significance Before/After Treatment****
(Adjusted p < 0.0001)		****
(Adjusted p < 0.0001)		-
Average Removal Rate53.75%70.18%****
(Adjusted p < 0.0001)
TP
(mg/L)		0 h226.67 ± 8.51227.00 ± 7.00ns
(Adjusted p = 0.9999)
48 h108.67 ± 4.6274.33 ± 3.51***
(Adjusted p = 0.0007)
Significance Before/After Treatment****
(Adjusted p < 0.0001)		****
(Adjusted p < 0.0001)		-
Average Removal Rate53.30%67.40%****
(Adjusted p < 0.0001)
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MDPI and ACS Style

Liu, J.; Yan, X.; Ding, Q.; Xiang, J.; Wei, Z.; Yang, Q.; Xie, K.; Cheng, B.; Xie, X. Porous Carbon Derived from Pumpkin Tissue as an Efficient Bioanode Toward Wastewater Treatment in Microbial Fuel Cells. Sustainability 2025, 17, 4758. https://doi.org/10.3390/su17114758

AMA Style

Liu J, Yan X, Ding Q, Xiang J, Wei Z, Yang Q, Xie K, Cheng B, Xie X. Porous Carbon Derived from Pumpkin Tissue as an Efficient Bioanode Toward Wastewater Treatment in Microbial Fuel Cells. Sustainability. 2025; 17(11):4758. https://doi.org/10.3390/su17114758

Chicago/Turabian Style

Liu, Jiaxin, Xue Yan, Qiang Ding, Jiwu Xiang, Zuna Wei, Qian Yang, Kangwei Xie, Bo Cheng, and Xiaoying Xie. 2025. "Porous Carbon Derived from Pumpkin Tissue as an Efficient Bioanode Toward Wastewater Treatment in Microbial Fuel Cells" Sustainability 17, no. 11: 4758. https://doi.org/10.3390/su17114758

APA Style

Liu, J., Yan, X., Ding, Q., Xiang, J., Wei, Z., Yang, Q., Xie, K., Cheng, B., & Xie, X. (2025). Porous Carbon Derived from Pumpkin Tissue as an Efficient Bioanode Toward Wastewater Treatment in Microbial Fuel Cells. Sustainability, 17(11), 4758. https://doi.org/10.3390/su17114758

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