Design of electroactive consortium synthesizing flora enables synergistic biomass-to-bioenergy conversion

Summary

Efficient biomass-to-energy conversion is key to sustainable development. We engineered a dual-functional synthetic microbial communities(SMC) (Bacillus subtilis XJU-1, XJU-2, Bacillus cereus XJU-3, and Enterobacter hormaechei XJU-5) to simultaneously degrade marine biomass and generate electricity in microbial fuel cells (MFCs). Using porous nickel-molybdenum foam as the anode, the 2-L MFCs achieved 941.67 mV and 443.67 mW m⁻³. Multi-omics analyses showed that vitamin B₆ and amino acid pathways boosted NADH/FADH₂ production, while higher SDH and COX activities enhanced electron transfer. The community converted 77%–90% of seven marine substrates into soluble nutrients, which correlated with increased power output. Four MFCs connected in series delivered 2187.17 mV and 598.13 mW m⁻³, and a 28-L MFCs generated stably for 30 days. Effluents rich in nitrogen, phosphorus, and potassium suggest potential use as diluted liquid fertilizers, supporting integrated bioenergy production and resource recycling.

Graphical abstract

Highlights

• •Engineered dual-functional SMC enabling biomass degradation and electricity generation
• •Achieved 941.67 mV and 443.67 mW m⁻³ using porous porous nickel-molybdenum foam anodes in 2 L MFCs
• •Linked vitamin B₆-amino acid metabolism to enhanced NADH/FADH₂ supply and transfer
• •Scaling to 28 L extended discharge to 30 days and boosted voltage by ∼3.4-fold

Biological sciences; Bioengineering; Biotechnology

Introduction

Efficient conversion of biomass waste into renewable energy is a key strategy for alleviating energy shortages, reducing greenhouse gas emissions, and mitigating environmental pollution. Conventional biomass-to-energy technologies, including anaerobic digestion, biogas fermentation, and bioethanol production, have been widely implemented in the renewable energy sector.^1,2,3 Anaerobic digestion employs methanogenic microorganisms to convert agricultural residues, sludge, or manure into biogas, enabling chemical energy recovery; however, it generates residual solids and wastewater that require further treatment.⁴ Bioethanol fermentation, using microbes such as yeast, converts starch- or sugar-rich substrates into ethanol as a fossil fuel substitute, yet it is constrained by substrate type and produces CO₂ and fermentation residues.⁵ These approaches generally exhibit low substrate adaptability and are inefficient in processing complex biomass, nitrogen-rich waste, or high-salinity materials, often resulting in secondary pollution.

In contrast, MFCs based on SMC exploit the synergistic functions of multifunctional microbes to efficiently degrade complex substrates and directly transfer electrons to electrodes, enabling direct conversion of biomass energy into electricity while reducing environmental impact.⁶ Biomass resources are highly diverse, including agricultural residues, municipal organic waste, and marine biomass, among others, with substantial variations in their composition and energy potential.^7,8,9 Agricultural and forestry residues are rich in cellulose and hemicellulose, providing stable and abundant sources, but they require energy-intensive pretreatment and exhibit slow degradation rates.¹⁰ Municipal sludge and food waste contain high levels of lipids and carbohydrates, allowing rapid conversion to methane or ethanol; however, their complex composition increases treatment costs.¹¹ By comparison, marine biomass and fishery byproducts present distinct advantages.^12,13 They are abundant—annual aquaculture processing alone generates millions of tons of discarded fish tissues, scales, and viscera, which are rich in proteins, amino acids, and biodegradable organic nitrogen, offering high-quality substrates for microbial metabolism and electron transfer.¹⁴ Moreover, marine biomass typically exists under high-salinity and high-organic-load conditions, providing an ideal model for evaluating the stability and electron transfer capacity of SMC under extreme environmental conditions.

Despite the potential of MFCs for biomass treatment, several critical bottlenecks limit their practical application. First, many electrogenic bacteria, such as Shewanella and Geobacter sulfurreducens, cannot directly degrade complex biomass and rely on non-electrogenic microbes to convert it into simpler substrates, preventing simultaneous degradation and electricity generation.^15,16 Second, imbalances within synthetic microbial consortia or insufficient degradation rates can compromise overall power output.¹⁷ For example, although cellulolytic consortia can convert steam-exploded corn stover solids into electricity, their power output remains significantly lower than that of the corresponding liquid fractions.^17,18 The low electrical conductivity of biomass hydrolysates severely limits their potential for large-scale electricity generation, with a 12 m³ microbial fuel cell treating brewery wastewater producing only 8 W/m³.¹⁹ In addition, the structural properties and surface area of anode materials critically influence microbial attachment and conductivity, thereby limiting electricity output.²⁰ Finally, insufficient environmental robustness renders MFCs prone to inactivation under high salinity, elevated pH, or complex wastewater conditions.²¹ Therefore, advances in microbial community engineering, electrode design, and system stability regulation are essential to realize the full potential of MFCs-based biomass treatment.

In this study, we systematically analyzed the electricity-generating performance of an SMC previously constructed in our laboratory for the degradation of terrestrial animal carcasses.²² Building on this foundation, we engineered a dual-functional SMC capable of both biomass degradation and electricity production, and implemented it in a MFCs system to achieve efficient coupling of marine biomass decomposition and bioelectricity generation. By evaluating different anode materials and surface areas, we identified the optimal configuration for this system. Integrating genome annotation with metabolomic analyses, we elucidated the mechanisms underlying key strains’ degradative and electroactive functions. To address the limitations of MFCs, such as low power density and reactor scale constraints, we conducted tandem and scale-up experiments and assessed the potential application of the post-MFCs effluent as fertilizer. Collectively, this work aims to enhance MFCs performance and biomass resource utilization, providing new technological strategies for sustainable energy development and environmental remediation.

Results

Construction of the MFCs device

Scanning electron microscopy showed distinct surface morphologies of carbon cloth, carbon cloth doped with carbon nanotubes, porous nickel foam mesh, and porous nickel-molybdenum foam (Figures 1A–1D). After operation, the highest microbial attachment was observed on the porous nickel-molybdenum foam electrode (Figures 1E–1H), which yielded an output voltage of 829 mV (Figure 1K). Under identical conditions, porous nickel-molybdenum foam, porous nickel foam mesh, carbon cloth doped with carbon nanotubes, and carbon cloth electrodes all showed a pair of redox peaks around −0.25 V, corresponding to the reversible Ni²⁺/Ni³⁺ couple (Figure 1I). The porous nickel-molybdenum foam electrode displayed the highest and most symmetric peaks, indicating the fastest electron transfer and strongest reversibility, followed by porous nickel foam mesh. Carbon cloth doped with carbon nanotubes and carbon cloth exhibited weaker current responses, suggesting lower electrochemical activity. Consistent with these CV results, EIS analysis further confirmed that porous nickel-molybdenum foam possessed the smallest charge-transfer resistance (Rct = 16.14 Ω), significantly lower than the other electrodes (Figure 1J). This minimal Rct demonstrates that Mo incorporation markedly accelerates interfacial charge transfer and increases the density of electrochemically active sites, thereby substantially enhancing the power-generation performance of MFCs. Polarization and power density analyses demonstrated maximum power densities of 268.33 mW m⁻³ (537 mV), 315.50 mW m⁻³ (631 mV), 378.83 mW m⁻³ (758 mV), and 414.33 mW m⁻³ (829 mV) for carbon cloth, carbon cloth doped with carbon nanotubes, porous nickel foam mesh, and porous nickel-molybdenum foam, respectively (Figure 1L). Surface area evaluation of nickel-molybdenum foam revealed that a 25 cm² electrode achieved a 2.550-fold higher voltage than 2.5 cm², while 37.55 cm² reached 2.553-fold; further enlargement caused a decline to 22.534-fold (Figure 1M), with power density following a similar trend (Figure 1N). Based on these results, porous nickel-molybdenum foam with a surface area of 25 cm² was selected for subsequent experiments.

Figure 1.

Characterization of different anode materials for MFCs

(A–D) Structures of the four anode materials: (A) carbon cloth (Scale bars: 5 μm), (B) carbon cloth doped with carbon nanotubes (Scale bars: 5 μm), (C) porous nickel foam mesh (Scale bars: 1 mm), and (D) porous nickel–molybdenum foam (Scale bars: 1 mm).

(E–H) Corresponding electrode surface colonization by microbial communities (Scale bars: 5 μm (E–F), 10 μm (G–H)).

(I) CV-based characterization of four anode materials in MFCs.

(J) EIS analysis of four anode materials in MFCs. (Fitted results (dots) and measured data (solid lines)).

(K) Output voltages of MFCs with different anode materials.

(L) Polarization and power density curves of MFCs with different anode materials.

(M) Output voltage of MFCs using porous nickel-molybdenum anodes with varying surface areas.

(N) Polarization and power density curves of MFCs with porous nickel-molybdenum anodes of varying surface areas.

Building synthetic microbial communities

Among the five degradative strains, each exhibited distinct enzymatic advantages: B. subtilis XJU-2 showed the highest protease activity, while E. hormaechei XJU-5 exhibited relatively high keratinase activity. These complementary enzymatic profiles enabled synergistic degradation among the strains (Figure 2B). In terms of electricity-generating performance, from day 7 onward (Figures 2C and 2D), P. megaterium XJU-4 exhibited a marked decrease in output voltage, accompanied by a decline in power density, indicating its poor electroactive performance. Plate co-culture experiments and growth curve analyses indicated that the synthetic microbial community exhibited robust growth, with individual strains capable of synergistic coexistence (Figure S1). Ultimately, an SMC comprising B. subtilis XJU-1, B. subtilis XJU-2, B. cereus XJU-3, and E. hormaechei XJU-5, which possesses dual functions of biomass degradation and electricity production, was successfully established.

Figure 2.

Analysis of strain performance and electrogenic mechanism

(A) Mechanism of electrogenesis in SMC.

(B) Enzyme activities of protease, lipase, and keratinase produced by the strains. (Data are presented as mean ± s.d. from n = 3 biological replicates per condition.).

(C) Output voltage generated by different bacterial strains in MFCs.

(D) Polarization and power density curves of different bacterial strains used in MFCs.

(E) Statistics of genes associated with cellular respiration and electron transport in bacterial strains.

(F) Succinate dehydrogenase activity in single strains and SMC. (All statistical analyses were performed using Dunnett’s test. Data are presented as mean ± s.d. with n = 3 biological replicates per condition. Significance is indicated as follows: ^nsp ≥ 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.).

(G) Cytochrome c oxidase activity profiles of individual strains and SMC. (All statistical analyses were performed using Dunnett’s test. Data are presented as mean ± s.d. with n = 3 biological replicates per condition. Significance is indicated as follows: ^nsp ≥ 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.).

Respiratory chain and electron transport gene analysis

Key respiratory chain enzyme activities differed significantly among individual strains and the SMC. Succinate dehydrogenase (SDH) activity was comparable among B. subtilis XJU-1, XJU-2, and B. cereus XJU-3, but was significantly lower in E. hormaechei XJU-5 (p < 0.05), while the SMC exhibited the highest SDH activity (Figure 2F). Cytochrome c oxidase (COX) activity was lowest in XJU-1 (∗∗p < 0.001) and significantly higher in XJU-2 and XJU-5 (p < 0.05, ∗∗p < 0.001), with the SMC showing the highest activity (1.5–2× single strains) (Figure 2G). These results indicate that metabolic complementarity and synergistic electron transfer among strains enhance overall energy metabolism, promoting electron release and improving MFCs electricity generation.

Genomic analysis supported these findings: the SMC harbored the largest number of key respiratory genes, including F₀F₁-type ATP synthase (36), terminal cytochrome oxidase (22), and cytochrome c biogenesis genes (21), correlating with enhanced enzyme activities and superior electrogenic performance (Figure 2E).

Metabolome analysis of synthetic microbial communities

Microorganisms can interact with one another through intermediate metabolites, forming complex metabolic networks that enable them to function collaboratively. Metabolomic profiling of the SMC (B. subtilis XJU-1, B. subtilis XJU-2, B. cereus XJU-3, and E. hormaechei XJU-5) revealed distinct differences compared with single-strain cultures. PCA analysis revealed clear separation between groups and minimal within-group variation, indicating good reproducibility and distinct group-specific characteristics (Figure 3A). Volcano plots demonstrated significant upregulation of multiple metabolites in the synthetic population (Figures 3B–3E). Key metabolites included N-formyl-L-methionine, L-carnitine, and 3-methylxanthine. KEGG enrichment analysis indicated that these differential metabolites were mainly associated with five pathways: protein digestion and absorption, D-amino acid metabolism, aminoacyl-tRNA biosynthesis, histidine metabolism, and tyrosine metabolism (Figure 3F).

Figure 3.

Metabolite analysis of secondary differences in synthetic microbial communities (SMC)

(A) Principal-component analysis (PCA) of single bacterial strains and SMC.

(B) Volcano plot of XJU-1 vs. SMC.

(C) Volcano plot of XJU-2 vs. SMC.

(D) Volcano plot of XJU-3 vs. SMC.

(E) Volcano plot of XJU-5 vs. SMC.

(F) KEGG analysis of metabolites of secondary differences in SMC.

Degradation of biomass using synthetic microbial communities

The degradation experiments involving seven types of biomass—namely, butterfish, corvinas, squid, hairtail, flatfish, bombay, and codfish—were conducted in a 3 L bioreactor (Figure 4A). The degradation of these biomasses was facilitated by the dynamics and metabolism of microorganisms within SMC. After 108 h of degradation, the biomass transitioned from a solid to a liquid state, with the final residual degradation product primarily consisting of calcium carbonate, which is relatively stable in nature. The microorganisms involved in this degradation process lacked the necessary enzyme systems or metabolic pathways to further degrade and utilize this residue. The degradation rate achieved through this method ranged from 77% to 90%, with varying rates for different biomasses, primarily influenced by the fish bone content of each biomass. During the degradation process, varying thicknesses of foam formed on the surface of the biomass degradation solution, and differences in the coloration of the degradation solutions were observed.

Figure 4.

Degradation of biomass with synthetic microbial communities

(A) Biomass degradation.

(B) Analysis of biomass degradation fluid composition. (The data are represented as mean ± s.d. with n = 3 biological replicates per condition.).

(C and D) Electricity production from biomass degradation fluids: (C) Output voltage of MFCs with different biomass anodes. (D) Polarization and power density curves of MFCs with different biomass anodes.

Tests on the electrical performance of biomass degradation fluids

Analysis of small molecule concentrations in biomass degradation solutions and their corresponding MFCs output (Figures 4B–4D) showed that solutions with higher electrochemical performance were enriched in triglycerides, amino acids (particularly branched-chain amino acids), and proteins. The highest output voltage and power density were observed for Corvina at 941.93 mV and 443.92 mW m⁻³, respectively, with concentrations of triglycerides, total amino acids, branched-chain amino acids, and proteins at 348.4 μM, 59.3 μM, 8.9 μM, and 2.1 mg/mL. In contrast, no triglycerides were detected in the squid and flatfish degradates, and the concentrations of amino acids, branched-chain amino acids, and proteins in squid and hairtail degradates were markedly lower than those in other samples, correlating with reduced MFCs output. Reducing sugar concentrations were relatively consistent among all solutions, with cod showing a slightly higher level of 0.25 mg/mL, but these differences did not significantly affect output voltage in this experiment.

Metabolomic analysis of biomass degradation fluids

Metabolomic analysis of the three sample groups identified 1,554 distinct metabolites belonging to 101 taxa, with 17 taxa containing more than 10 metabolites. PCA showed distinct, reproducible group separation with minimal within-group variation (Figure 5A). The most abundant classes were benzene and its substituted derivatives, carboxylic acids and their derivatives, fatty acyls, and organooxygen compounds (Figure 5B). Heatmap analysis revealed distinct metabolite distributions among the three groups (Figure 5C). Group C showed elevated levels of indole-3-carboxaldehyde and 5-pyridoxolactone; group D was characterized by 4alpha,5alpha-epoxy-11-eudesmen-3a-ol and isoglutamine; and group E exhibited high levels of 2-methyl-1,5-dinitro-3-nitrosobenzene,4-Hydroxy-3-methoxybenzenemethanol coniferyl alcohol, and so on. Aromatic compounds and derivatives, such as 2-methyl-1,5-dinitro-3-nitrosobenzene and 4-hydroxy-3-methoxybenzenemethanol, facilitate microbial-electrode electron transfer through redox activity and chemical signaling, while coniferyl alcohol, shikonin, and 3,7-dimethylquercetin act as metabolic intermediates that modulate microbial metabolism or suppress competitors, indirectly enhancing electricity generation.^23,24 The KEGG pathway enrichment analysis demonstrated that the differential metabolites were primarily enriched in vitamin B6 metabolism, tryptophan metabolism, and alanine, aspartate, and glutamate metabolism (Figure 5D).

Figure 5.

Metabolomic analysis of biomass degradation fluids before and after electricity production

(A) Principal-component analysis (PCA) of biomass degradation fluids.

(B) Histogram illustrating the relative abundance of metabolite taxa.

(C) Heatmap illustrating significant differences.

(D) Metabolic pathway enrichment analysis.

Series and amplified MFCs to improve power production performance

Polarization curves were obtained by varying the external resistance from 10 to 1000 Ω, with the power density reaching 973.84 mW m⁻³ at 200 Ω, corresponding to the system’s optimal load (Figure S2). However, to evaluate operational stability, the MFCs performance was continuously monitored under an external resistance of 1000 Ω, close to the open-circuit voltage, yielding a maximum output voltage of 941.67 mV and a power density of 443.67 mW m⁻³. When two MFCs were connected in series, the maximum output voltage and power density increased to 1458.23 mV and 531.81 mW m⁻³, while four MFCs in series produced 2187.17 mV and 598.13 mW m⁻³ (Figures 6C and 6D). The open-circuit voltage of four MFCs in series reached 4.7 V, sufficient to power 2.2–2.4 V LEDs (Figure 6A). Scaling up the MFCs device from 2 L to 28 L increased the maximum output voltage from 338.00 mV to 1145.10 mV but decreased the maximum power density from 57.12 mW m⁻³ to 46.83 mW m⁻³ (Figures 6E and 6F). The enlarged system extended the self-sustained power generation period from 20 to 30 days.

Figure 6.

MFCs series and amplification experiments

(A) 4 series MFCs devices powering LED light-emitting diodes.

(B) Design and test diagrams for the 28 L MFCs.

(C) Output voltages of MFCs with different series connection configurations.

(D) Polarization and power density curves of MFCs with different series connection configurations.

(E) Output voltages of 28 L and 2 L MFCs devices.

(F) Polarization and power density curves of 28 L and 2 L MFCs devices.

Reuse of marine biomass degradate resources

The nutrient composition of seven mixed biomass degradation liquids after power generation was analyzed, revealing the presence of both macro- and micro-elements. The concentrations of calcium (Ca), magnesium (Mg), and sulfur (S) exceeded the requirements specified in the standards of the Ministry of Agriculture and Rural Development of the People’s Republic of China for the implementation of elements of water-soluble fertilizers (NY/T 1107–2020) and the Michigan Department of Agriculture and Rural Development’s Label Requirements of Specialty and other bagged fertilizers, thereby meeting the criteria for liquid fertilizers. In contrast, the concentrations of total nitrogen (TN), total phosphorus (TP), and potassium (K) were significantly higher than the upper limits of the standards, while copper (Cu), iron (Fe), and manganese (Mn) were below the required levels (Table 1).

Table 1.

Determination of plant nutrient content of biomass degradation solution

Class	Elements	Concentration (mg/mL)	Liquid fertilizer standard (mg/mL)
			America	China
Major elements	TN	3890.000 ± 40.000	120	≥40
	TP	334.333 ± 3.512	150	≥40
	K	789.667 ± 9.504	240	≥40
Middle element	Ca	902.000 ± 2.000	≥10	≥1
	Mg	580.000 ± 2.013	≥5	≥1
	S	773.000 ± 12.000	≥10	≥1
Trace element	B	2.423 ± 0.006	≥0.2	≥0.5
	Cu	0.050 ± 0.002	≥0.5	≥0.5
	Fe	0.430 ± 0.056	≥1	≥0.5
	Mn	0.070 ± 0.002	≥0.5	≥0.5
	Zn	0.500 ± 0.023	≥0.5	≥0.5

Discussion

This study demonstrates that an SMC composed of B. subtilis XJU-1, XJU-2, B. cereus XJU-3, and E. hormaechei XJU-5 can simultaneously achieve marine biomass degradation and electricity generation in MFCs systems. Comparison of different anode materials revealed that a porous nickel-molybdenum foam anode performed best, yielding a maximum output voltage of 829 mV and a power density of 414.33 mW m⁻³ in a 2 L MFCs with a 1 kΩ external resistor in series. Integrated analyses of gene annotation, enzyme activity, and metabolomics indicated that the consortium enhances central carbon metabolism by modulating vitamin B₆ and amino acid pathways, thereby promoting NADH and FADH₂ production. Concurrently, increased activities of SDH and COX further optimized electron transfer and proton translocation (Figures 2A, 2E, 2F, 2G, 3F, and 5D). When four MFCs units were operated in series, the output voltage and power density increased to 2187.17 mV and 598.13 mW m⁻³, respectively; scaling up to a 28 L system extended the discharge duration from 20 to 30 days and increased the output voltage by approximately 3.4-fold. These findings suggest that the synergy between microbial metabolism and electrode properties is critical for enhancing electricity generation, while also providing a theoretical basis for the scale-up of MFCs technology and the development of biomass degradation effluents as nutrient-rich fertilizers.

Previous studies have commonly employed single strains or natural microbial consortia in MFCs for organic substrate degradation and electricity generation; however, these systems often suffer from limited electron transfer efficiency, unstable power output, and incomplete biomass degradation due to heterogeneous microbial functions and insufficient electrode-microbe interfaces.^25,26,27 In this study, we established an SMC coupled with a porous nickel-molybdenum foam anode to achieve efficient integration of marine biomass degradation and bioelectricity production. The Ni-Mo anode provided a highly conductive and porous surface that facilitated biofilm formation and strengthened microbe-electrode interactions, thereby enhancing extracellular electron transfer (EET).^28,29 Multi-omics and enzyme activity analyses revealed that the consortium optimized intracellular energy metabolism through the coordinated regulation of vitamin B₆ and amino acid pathways, which activated glycolysis and the tricarboxylic acid cycle to increase NADH and FADH₂ production.³⁰ Elevated activities of SDH and COX further accelerated electron flow along the respiratory chain and improved proton translocation efficiency.³¹ Notably, several metabolites such as adenine, 3′-AMP, flavins, and quinones acted as endogenous redox mediators to facilitate bidirectional electron transfer between microbes and electrodes, while 3-methylxanthine and ergocalciferol helped alleviate oxidative stress and maintain community stability.^32,33 Together, these results indicate that the synergistic design of the porous nickel-molybdenum foam anode and the SMC not only enhances microbial adhesion and EET capacity but also restructures the intracellular metabolic network toward efficient energy coupling.

During the scale-up process, although a 28 L MFCs maintained a relatively high output voltage, its volumetric power density was significantly lower than that of the laboratory-scale system. This discrepancy is possibly attributed to mass transfer limitations in the larger system, where the diffusion efficiency of substrates and metabolic products at the electrode surface is reduced; the decreased electrode surface-to-volume ratio restricts microbial attachment and biofilm development; and the increased internal resistance diminishes electron transfer efficiency.^34,35,36,37 Previous studies have proposed various engineering strategies to address these challenges, such as employing three-dimensional porous or stacked electrode architectures to enhance effective surface area, implementing recirculating feeding and hydrodynamic optimization to mitigate mass transfer barriers, and adopting modular electrode designs to reduce system internal resistance.^38,39 For large-scale MFCs, optimizing electrodes, improving mass transfer, and reducing internal resistance can enhance power density and stability.

In terms of agricultural utilization, the nutrient composition of the degradation effluent exhibits potential value.⁴⁰ Analytical results indicate that the effluent contains total nitrogen, total phosphorus, and potassium levels far exceeding conventional fertilizer standards, whereas trace elements, such as copper, iron, and manganese are relatively deficient. Direct application to farmland could therefore lead to nutrient overloading and trace element imbalances, adversely affecting crop growth and soil health. A rational strategy involves diluting the effluent to reduce N, P, K concentrations while supplementing essential trace elements such as iron and manganese to ensure crop nutritional balance. This “dilution plus supplementation” approach not only promotes the resource-efficient use of degradation effluent but also enhances the feasibility of integrating MFCs technology within agricultural circular systems.

This study demonstrates that an SMC composed of B. subtilis XJU-1, XJU-2, B. cereus XJU-3, and E. hormaechei XJU-5 can efficiently couple marine biomass degradation with electricity generation in MFCs. A porous nickel-molybdenum foam anode showed the best performance, and integrated analyses of gene annotation, enzyme activity, and metabolomics revealed that the consortium enhances central carbon metabolism via vitamin B₆ and amino acid pathways, while succinate dehydrogenase and cytochrome c oxidase improve electron transfer efficiency. Series and scale-up operation further increased voltage, power density, and discharge duration. Previous studies indicated that this consortium also effectively degrades terrestrial biomass, highlighting its broad potential for biomass degradation and electricity generation in both marine and terrestrial contexts.²² These findings underscore that the synergy between microbial metabolism and electrode performance is critical for enhancing electricity generation and provide a theoretical basis for MFCs scale-up and the development of biomass degradation effluents as nutrient-rich fertilizers.

Limitations of the study

While this study demonstrates the synergistic effects of electrode materials, SMC, and biomass substrates on MFCs performance, several limitations remain. First, the microbial consortium was constructed from a limited number of bacterial strains; additional species or naturally derived communities may further enhance degradation efficiency and electricity generation. Second, although scaling experiments up to 28 L provided insights into practical applications, further scale-up to pilot or industrial levels may encounter challenges related to mass transfer, electrode geometry, and system stability. Third, to evaluate the self-sustaining capability of the MFCs, this study conducted polarization curve measurements on the 2 L MFCs under different external resistances only at the optimal operating conditions. This approach may limit a comprehensive understanding of the system’s dynamic responses. Future studies should consider long-term operation and performance evaluation under a wider range of operational conditions. Finally, while metabolomic and genomic analyses provided mechanistic insights, in situ measurements of electron flux and real-time biofilm dynamics would further clarify the functional contributions of individual strains and metabolites. Addressing these limitations will be critical for advancing MFCs technology toward large-scale, sustainable bioenergy, and nutrient recovery applications.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Dr. Haitao Yue (yuehaitao@tsinghua.org.cn).

Materials availability

All the materials of this study are available from the lead contact without restriction upon request.

Data and code availability

• •All data reported in this article will be shared by the lead contact upon request.
• •No original code was generated for this study.
• •Additional information on the data reported in this article and their analysis is available from the lead contact on request.

Acknowledgments

This study is supported by Tianshan Young Top Talents-Basic Research Talents (2024TSYCJU0002), Key Research and Development Project of Xinjiang Uygur Autonomous Region of China (grant 2023B02034 and 2023B02034-2), The Third Xinjiang Scientific Expedition Program, National Key Research and Development Program of China (grant 2022xjkk020603) and National Natural Science Foundation of China (grant U2003305).

Author contributions

Writing – original draft, visualization, resources, investigation, N.W.; writing–original draft, validation, resources, investigation, Z.C.; validation, methodology, J.W.; writing–original draft, visualization, R.W.; resources, investigation, Y.L.; validation, Y.W.; validation, K.Z.; writing–review and editing, supervision, project administration, funding acquisition, H.Y.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial strains
Bacillus subtilis XJU-1	Chinese Center for the Conservation of Typical Cultures (CCTCC)	CCTCC No. M202304
Bacillus subtilis XJU-2	Chinese Center for the Conservation of Typical Cultures (CCTCC)	CCTCC No. M202305
Bacillus cereus XJU-3	Chinese General Microbial Strain Conservation and Management Center (CGMCC)	CGMCC No. 25397
Priestia megaterium XJU-4	Chinese Center for the Conservation of Typical Cultures (CCTCC)	CCTCC No. M2023309
Enterobacter hormaechei XJU-5	Chinese Center for the Conservation of Typical Cultures (CCTCC)	CCTCC No. M2023306
Chemicals
Tris–HCl	Servicebio Technology Co., Ltd.	Cat# GC208003
Sodium acetate (CH3COONa)	Beijing Solarbio Science & Technology Co., Ltd.	Cat# A1070
Ammonium chloride (NH4Cl)	Beijing Solarbio Science & Technology Co., Ltd.	Cat# 12125-02-9
Potassium Dihydrogen Phosphate (KH2PO4)	Beijing Solarbio Science & Technology Co., Ltd.	Cat# 7778-77-0
Dipotassium hydrogen phosphate (K2HPO4)	Beijing Solarbio Science & Technology Co., Ltd.	Cat# 7758-11-4
Magnesium chloride hexahydrate (MgCl2·6H2O)	Beijing Solarbio Science & Technology Co., Ltd.	Cat# 7791-18-6
Calcium chloride dihydrate (CaCl2·2H2O)	Beijing Solarbio Science & Technology Co., Ltd.	Cat# 10035-04-8
Potassium chloride (KCl)	Beijing Solarbio Science & Technology Co., Ltd.	Cat# 7447-40-7
Artificial sea water	Dalian Haibao Biotechnology Co., Ltd.	N/A
Cytochrome c Oxidase Activity Assay Kit	Beyotime Biotechnology Co., Ltd.	Cat# P0421S
Succinate Dehydrogenase Activity Assay Kit	Beyotime Biotechnology Co., Ltd.	Cat# S0530S
Amplex Red Triglyceride Assay Kit	Beyotime Biotechnology Co., Ltd.	Cat# S0219M
BCA Protein Assay Kit	Beyotime Biotechnology Co., Ltd.	Cat# P0012
Amino Acid (AA) Content Detection Kit	Sangon Biotech (Shanghai) Co., Ltd.	Cat# AK0418
Branched-Chain Amino Acid Detection Kit (WST-8 Method)	Beyotime Biotechnology Co., Ltd.	Cat# S0535S
Critical commercial assays
Metabolomic analysis	Suzhou Panomic Biomedical Technology Co., Ltd.	https://www.panomix.com/
Software and algorithms
ZView 3.3	AMETEK Scientific Instruments	https://www.ameteksi.com/
CHI660E	CH Instruments, Ins.	https://www.chinstruments.com
Origin 2026	OriginLab	https://www.originlab.com
R Language	The R Project for Statistical Computing	https://www.r-project.org/

Experimental model and study participant details

Strain and culture conditions

The experimental strains used in this study originated from our laboratory and have been deposited in both the Chinese Center for the Conservation of Typical Cultures (CCTCC) and the Chinese General Microbial Strain Conservation and Management Center (CGMCC). These strains include Bacillus subtilis XJU-1 and XJU-2, Bacillus cereus XJU-3, Priestia megaterium XJU-4, and Enterobacter hormaechei XJU-5, all of which were routinely cultured in LB liquid medium at 37 °C.

Method details

Analysis of cellular respiration and electron transport genes

Escherichia coli BL21 (Ec BL21) was used as the control, and genes associated with cellular respiration and electron transport in both electrogenic strains and synthetic electrogenic communities were identified and analyzed. Genome annotation and functional classification were performed using the RAST platform.⁴¹

Enzyme activity assays

Protease and lipase activities were determined by the Folin–Ciocalteu and olive oil flotation methods, respectively.²² Keratinolytic activity was measured by incubating 0.25 mL crude enzyme with 0.5 mL 0.05 M Tris–HCl (pH 7.5) and 5 mL keratin at 37 °C, 120 rpm for 2 h, stopping with 0.5 mL 10% TCA, centrifuging (10,000 g, 15 min), and reading the supernatant at 280 nm.⁴² Succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) activities in single strains and synthetic microbial communities were measured using Beyotime Succinate Dehydrogenase Activity Assay Kit (Colorimetric) and Beyotime Cytochrome c Oxidase Activity Assay Kit (Colorimetric), respectively, following the manufacturers’ instructions.

MFCs assembly

The MFC systems utilized in this study consist of a 2 L dual-chamber MFCs unit (purchased from Jinan North Glass Instrument Co.) and a custom-designed 28 L MFCs unit. The anode and cathode chambers of the 2 L MFCs are separated by a Nafion N117 proton exchange membrane and are connected by two graphite electrodes. The two electrodes were electrically connected through pure copper wires with a single 1 kΩ external resistor in series. This high-resistance connection allowed for continuous voltage monitoring while maintaining minimal current flow, thereby enabling the evaluation of the electrochemical and biological stability of the MFCs over time under quasi–open-circuit conditions. However, we also evaluated the power density of the MFCs under optimal conditions with different external resistances, using the selected anode material and marine biomass co-digestion effluent. In addition, the 28 L MFCs (Outer diameter (OD) = 30 cm,Height (h) = 40 cm) is also separated by a Nafion N117 proton exchange membrane and connected by two electrodes, which are linked by pure silver wires(The silver wires exhibit higher electrical conductivity than copper wires.⁴³) with a 1 kΩ resistor in series. The MFCs was operated in a 28 L reactor equipped with a 1131 cm² porous nickel-molybdenum foam anode wound along the inner wall and a 300 cm² carbon cloth cathode, maintaining an inter-electrode distance of 1 cm. Furthermore, to simulate the degradation of in situ marine biomass, the 28-L MFCs was evaluated with the 2-L MFCs serving as the control group, and artificial seawater was used as the catholyte.

Determination of MFCs anode electrode material and surface area

1000 mL of anode electrolyte was prepared, consisting of the following concentrations: 1.64 g/L CHCOONa, 0.5 g/L NH₄Cl, 4.4 g/L KH₂PO4, 3.4 g/L K₂HPO₄, 0.1 g/L MgCl₂·6H₂O, 0.1 g/L CaCl₂·2H₂O, and 0.1 g/L KCl. This solution was injected into the anode chamber of a 2 L MFCs. Additionally, 100 mL of strains, which had been incubated for 48 h, was collected. The bacteria were harvested by centrifugation at 5000 rpm and 4°C for 10 min and subsequently added to the anode chamber. The cathode chamber was filled with 1000 mL of a 10% ammonium persulfate solution.

The anode electrodes were constructed from carbon cloth, carbon cloth doped with carbon nanotubes, porous nickel foam mesh, and porous nickel-molybdenum foam, with dimensions of 25 × 10 mm, 25 × 25 mm, 25 × 50 mm, 25 × 75 mm, 25 × 100 mm, 25 × 150 mm, and 25 × 200 mm, respectively. The cathode electrodes consisted of 25 × 100 mm carbon cloth. The output voltage of MFCs was recorded every 24 h using a data acquisition card (ART Technology, DAM3159HB) to evaluate the performance of the MFCs anode electrode materials and their surface areas.

Cyclic voltammetry (CV) was performed on a CHI660E electrochemical workstation (Shanghai CH Instruments, China) using a conventional three-electrode system, with the prepared sample as the working electrode, platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the Huang et al.⁴⁴ The potential was scanned from −0.8 V to +0.2 V (vs. SCE) at a rate of 0.01 V s⁻¹ with two segments, a sampling interval of 0.01 V, a quiet time of 2 s, and a sensitivity of 0.001 A V⁻¹. All measurements were conducted at room temperature (∼25 °C) in freshly prepared electrolyte purged with high-purity nitrogen for at least 15 min before testing. Electrode diffusion kinetics were investigated using electrochemical impedance spectroscopy (EIS) with a scan rate of 10 mV/s over a frequency range of 0.1–1×10⁵ Hz. ZView 3.3 was used for equivalent circuit fitting.

Scanning electron microscope

Scanning electron microscopy (SEM) is a powerful technique employed to observe the microscopic morphology of materials and the structure of biofilms.⁴⁵ Before examining the morphology, structure, and characteristics of the anode biofilm, it is essential to pre-treat the biofilm. This process involves fixing the anode biofilm in a 2.5% glutaraldehyde solution at 4°C overnight, followed by washing the biofilm twice with PBS. Subsequently, the anode biofilm is dehydrated in a gradient manner using ethanol solutions of 30%, 50%, 70%, 80%, and 90%. Finally, the samples are freeze-dried using a freeze dryer. Once the samples are completely dried, they are ready for analysis using SEM.

Building synthetic microbial communities

1000 mL of anode electrolyte was injected into the 2 L anode chamber of MFCs, and 1000 mL of a 10% ammonium persulfate solution was injected into the cathode chamber. Subsequently, 100 mL aliquots of the strains B. subtilis XJU-1, B. subtilis XJU-2, B. cereus XJU-3, P. megaterium XJU-4, and E. hormaechei XJU-5, which had been incubated for 48 h, were centrifuged at 5,000 rpm and 4°C for 10 min. The collected microorganisms were then added to the anode chamber. The output voltage of the MFCs was recorded every 24 h using a data acquisition card. The strains were evaluated for their ability to generate electricity, and an SMC was constructed to couple biomass degradation with electricity production.

Biomass degradation

500 g of butterfish, corvinas, squid, hairtail, flatfish, bombay and codfish were weighed and placed into a 3 L bioreactor. Subsequently, 2 L of water was added to the reactor, maintaining a water-to-fish volume-to-mass ratio of 4:1. Bacteria, obtained through the centrifugation of synthetic microbial colonies cultured for 48 h, were introduced into the reactor at a mass ratio of 15% relative to the fish. The reactor was then placed in a constant-temperature oscillatory incubator set to 37°C and operated at 100 rpm. After 108 h, the degradation process was completed, and the degradation residue was filtered using gauze to calculate the degradation rate. Seven distinct biomass degradation solutions were obtained.

$$\text{Degradation}\text{rate}=\frac{\text{Amount}\text{of}\text{degraded}\text{material}}{\text{Material}\text{quantity}}\times 100\%$$

Analysis of the composition of biomass degradation fluids

Triglyceride concentration was measured using the Amplex Red Triglyceride Test Kit from Beyotime Biotechnology, following the manufacturer’s instructions. Absorbance was recorded at 570 nm using a microplate reader, and concentration was calculated from a standard curve. Amino acid concentration was determined with the Amino Acid (AA) Content Assay Kit from Sangon Biotech (Shanghai), also measuring absorbance at 570 nm. The concentration of branched chain amino acids was assessed using the Branched Chain Amino Acid Test Kit with WST-8 from Beyotime Biotechnology, with absorbance measured at 450 nm and calculated from a standard curve. Protein concentration was determined using the BCA Protein Assay Kit from Beyotime Biotechnology, with absorbance recorded at 562 nm and calculated from a standard curve. Reducing sugar concentration was measured using the 3,5-dinitrosalicylic acid (DNS) assay according to Du’s method,⁴⁶ with absorbance measured at 540 nm and calculated from a standard curve.

Evaluation of the electrical performance of biomass degradation fluids

Seven biomass degradation solutions were injected into the anode chamber of the 2 L MFCs as the electrolyte. These degradation solutions contained electroactive microorganisms, thereby eliminating the need for separate additions. Concurrently, a 10% ammonium persulfate solution was injected into the cathode chamber. To evaluate the electricity production performance, the output voltage of the MFCs was recorded every 24 h using a data acquisition system, and the MFCs was maintained at room temperature throughout the entire process.

Metabolomic analysis

To analyze the interactions within the SMC, a metabolomic analysis of B. subtilis XJU-1, B. subtilis XJU-2, B. cereus XJU-3, E. hormaechei XJU-5, and the SMC was conducted using whole-target metabolomics. Additionally, to thoroughly investigate the changes in metabolites before and after electricity production from the biomass degradation solution, whole-target metabolomic analysis was performed on three sample groups: the first group consisted of the synthetic microbial community cultured in LB liquid medium for 24 h (Group C); the second group comprised the biomass degradation solution (Group D); and the third group included the anodic electrolyte at the conclusion of the MFCs performance test (Group E).

Take 2 mL of each of the samples mentioned above and transfer them into a 2 mL centrifuge tube. Centrifuge the samples at 5,000 rpm for 10 min at 4°C to remove the supernatant. Next, transfer an exact volume of 750 μL of the supernatant to a new 2 mL centrifuge tube, and vortex it with 400 μL of methanol for 1 min. Subsequently, centrifuge the mixture at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to a new tube, concentrate it, and dry it. The sample is then dissolved in 150 μL of 80% aqueous methanol containing 4 ppm of 2-chloro-1-phenylalanine. Filter the solution through a 0.22 μm membrane and transfer it to a detection vial. Finally, send the sample to Suzhou Panomic Biomedical Technology Co., Ltd. for LC-MS analysis to obtain the list of substances for quantification.⁴⁷

Differential metabolites were identified through a combined strategy of univariate tests (FC > 1, p < 0.01, FDR<1) and multivariate analysis (OPLS-DA, VIP>1.5), followed by hierarchical clustering with the pheatmap package and functional pathway enrichment using MetaboAnalyst and KEGG Mapper, ensuring statistical significance, biological interpretability, and pathway-level insights.⁴⁸

MFC series experiment

The MFCs were designed to conduct a series of experiments by connecting two to four MFCs units in series, specifically by linking the positive terminal of one MFCs to the negative terminal of the subsequent MFCs. Once the series connection was established, the two groups of MFCs were treated as new MFCs units, with each connected to an external 1 kΩ resistor. During the experiment, the output voltage of the MFCs was recorded every 24 h using a data acquisition card, and the MFCs were maintained at room temperature throughout the duration of the experiment.

Determination of internal resistance in MFC

A digital multimeter (DT9205A+) is utilized to measure the open-circuit voltage (E) of the battery. Connect the multimeter directly to the positive and negative terminals of the battery. At this stage, the external circuit is open, and the voltage measured by the multimeter is E. Next, connect a known resistance value of the load resistor to create a closed loop in the external circuit of the battery. Use the multimeter once more to measure the voltage across the load resistor (U), and then apply the appropriate formula to calculate the internal resistance of the MFCs (r).

$$\mathrm{r}=\frac{\left(\mathrm{E}-\mathrm{U}\right)\mathrm{R}}{\mathrm{U}}$$

Calculation of electrical parameters

The performance of MFCs was evaluated by calculating the current (I, mA), current density (J, mA m⁻³), and power density (P, mW m⁻³), all normalized to the effective volume of the system. The current was measured directly using a multimeter, and calculated based on Ohm’s law.

$$I=\frac{\mathrm{U}}{\mathrm{R}}$$

where U is the measured voltage (V or mV) and R is the external resistance (Ω). Current density was calculated by normalizing the measured current to the effective volume of the MFCs.

$$J=\frac{I}{V}$$

where V is the effective volume of the MFCs (m³). Power density (P) was determined as the product of the measured voltage and current, normalized to the effective volume of the MFCs.

$$P=\frac{U\ast I}{V}$$

Determination of plant nutrient content of biomass degradation solution

Determine the total nitrogen content in the biomass degradation solution at the end of power generation according to the standard HJ 636–2012, established by the Ministry of Ecology and Environment of the People’s Republic of China. Additionally, assess the total phosphorus content in accordance with the provisions of standard GB 11893-89, and evaluate the levels of potassium, calcium, magnesium, sulfur, boron, iron, manganese, zinc, and copper based on standard HJ 776–2015.

Quantification and statistical analysis

Statistical and correlation analyses were performed using Origin 2026 and R Language. All statistical analyses were performed using Dunnett’s test. Data are presented as mean ± s.d. with n = 3 biological replicates per condition. Significance levels are indicated as follows: ^nsP ≥ 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.

Published: December 17, 2025