Method for driving biochar and nanometer magnetite to synergistically strengthen anaerobic digestion of silage feed waste under weak magnetic field

Abstract

This invention discloses a method for synergistically enhancing the anaerobic digestion of silage waste using biochar and nano-magnetite driven by a weak magnetic field. This method, through the combined application of a weak magnetic field, nano-ferric oxide, and biochar, increases methane production by 67.7% during the anaerobic digestion of silage waste, enhances acetic acid production, and accelerates the conversion of acetic acid to methane. Under the drive of the weak magnetic field, the conductive network formed by biochar and nano-ferric oxide synergistically reduces the charge transfer impedance of the system, enhances the Fe(III) / Fe(II) redox cycle and interspecies direct electron transfer, and simultaneously increases the activity of key enzymes such as acetate kinase. This promotes the formation of a tighter metabolic interaction network between bacteria and archaea, ultimately achieving efficient coupling of carbon, electron, and energy flows within the anaerobic digestion system. This provides a promising technical strategy for improving the resource utilization efficiency of organic waste.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection technology, and particularly relates to the field of anaerobic treatment technology of silage waste in solid waste management. Specifically, it relates to a method for synergistic enhancement of anaerobic digestion of silage waste by using biochar and nano-magnetite driven by a weak magnetic field. Background Technology

[0002] In recent years, with economic development and the increasing demand for livestock products such as meat, eggs, and dairy products, silage has become an important means of ensuring the supply of forage for ruminants and promoting the sustainable development of animal husbandry, effectively making up for the shortage of fresh forage. According to data from the Food and Agriculture Organization of the United Nations (FAOSTAT) and the World Cereals Organization, global corn silage production is estimated to be between 360 million and 480 million tons. During the air-permeable storage and feeding stages, improper management can easily lead to the generation of spoiled silage waste (SW), which accounts for more than 10% of the total silage production. This waste is typically dark brown, viscous, and emits a foul odor. It easily produces acidic leachate and contains molds and pathogens (such as Listeria and Aspergillus), posing a significant risk of transmission and contamination. Long-term accumulation can pollute the air and groundwater, threatening the ecological environment of pastoral areas. Therefore, proper disposal of silage waste is a key link in protecting the ecological environment of pastoral areas and promoting the sustainable development of animal husbandry.

[0003] Silage, as an organic waste, can be treated through composting, biochar utilization, and anaerobic digestion (AD). Composting organic waste can reduce its volume by approximately 40%–60%. However, the composting process is affected by various factors such as temperature, moisture content, and carbon-to-nitrogen ratio. Improper control can easily lead to incomplete fermentation, insufficient heating, or excessive drying, prolonging the composting period or causing unstable product quality. Furthermore, in the initial stages of composting, the decomposition and ammoniation processes of organic matter produce large amounts of harmful gases such as ammonia and hydrogen sulfide (H2S). Meanwhile, the efficient production of biochar from organic waste requires high temperatures of 300–700°C under anaerobic or anoxic conditions, and faces challenges such as high cost of carbonization equipment and high start-up energy consumption. Anaerobic digestion, as one of the most promising treatment technologies, can achieve the harmless and volume-reduced treatment of silage waste while producing renewable energy sources such as methane, hydrogen, and ethanol. Liao et al. conducted a systematic study on 239 samples of poorly fermented silage (waste) and found that these samples had high potential for biogas and methanogenesis in an anaerobic digestion environment. The potential for developing bioenergy from silage waste is enormous. Although anaerobic digestion plays a crucial role in the resource utilization of organic waste, its system still has inherent limitations, especially the insufficient efficiency of interspecies electron transport kinetics. Electron transport by diffuse electron carriers such as hydrogen and formic acid is limited by mass transfer, thus restricting the metabolic rate of the anaerobic digestion process. Introducing conductive materials into the anaerobic digestion system is an effective means to promote direct interspecies electron transport (DIET) and improve methanogenesis efficiency. Among them, biochar (BC) and nano-ferric oxide (NF) have outstanding performance in promoting DIET due to their excellent conductivity and biocompatibility. Biochar tends to provide attachment sites for functional microorganisms, forming a dense biofilm network, while nano-ferric oxide is more likely to serve as an electron channel to enrich electroactive microorganisms. For example, adding biochar can enhance the expression of conductive pili (e-pili), regulate the microbial interaction network, strengthen interspecies direct electron transport, and promote the synergistic effect between anabolic bacteria and methanogens; adding 12-18 nm nano-ferric oxide to the anaerobic digestion system of urban sewage sludge can stimulate Fe 2+ The release of biochar accelerates potential interspecies direct electron transfer, increasing maximum gas production (Rmax) and cumulative methanogenic potential (M0), while simultaneously improving the removal rates of volatile suspended solids (VSS) and total chemical oxygen demand (TCOD) by approximately 26% and 35%, respectively. Based on the above research, the combined use of biochar and nano-ferric oxide can leverage the complementary advantages of microbial aggregation and electron transfer, potentially producing a synergistic enhancement effect.

[0004] Magnetic fields can regulate anaerobic digestion and promote methane production. Weak magnetic fields can activate microbial metabolism, enhance the interaction between metal ions and key enzymes, and improve the biodegradability of organic matter, showing significant enhancement potential. Magnetic fields can alter the structure of soil microbial communities; the abundance of methanogenic archaea increases after the application of a magnetic field. However, research on whether the functional combination of biochar and nano-ferric oxide can regulate the interspecies electron transfer efficiency of microbial communities in anaerobic digestion systems under weak magnetic field conditions, thereby achieving efficient methane production, is still limited.

[0005] This invention utilizes the synergistic effects of weak magnetic fields (WMF), biochar (BC), and nano-ferric oxide (NF) in an anaerobic digestion system for silage waste. By analyzing the degradation and transformation of organic matter, cumulative methane production, microbial electrochemical activity, microbial community structure and interaction networks, and predicting the responses of key nodes in metabolic pathways, this invention further elucidates the mechanisms by which these factors regulate interspecies electron transport pathways and efficiency, and enhance methane generation. Furthermore, this invention provides new insights into the role of direct interspecies electron transport in the interaction between weak magnetic fields, conductive materials (nano-ferric oxide and biochar), microorganisms, and organic waste (silage waste) in an anaerobic digestion system. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for synergistic enhancement of anaerobic digestion of silage waste using biochar and nano-magnetite driven by a weak magnetic field. This method, through the combined application of a weak magnetic field, nano-ferric oxide, and biochar, increases methane production by 67.7% during anaerobic digestion of silage waste, enhances acetic acid production, and accelerates the conversion of acetic acid to methane. Under the drive of the weak magnetic field, the conductive network formed by biochar and nano-ferric oxide synergistically reduces the charge transfer impedance of the system, enhances the Fe(III) / Fe(II) redox cycle and interspecies direct electron transfer, and simultaneously increases the activity of key enzymes such as acetate kinase. This promotes the formation of a closer metabolic interaction network between bacteria and archaea, ultimately achieving efficient coupling of carbon, electron, and energy flows within the anaerobic digestion system. This provides a promising technical strategy for improving the resource utilization efficiency of organic waste.

[0007] To achieve the above technical effects, the following technical solution is adopted: A method for synergistic enhancement of anaerobic digestion of silage waste using a weak magnetic field-driven biochar and nano-magnetite is as follows: In a reactor equipped with a single-valve gas collection bag, silage waste is added, sludge is inoculated, and ultrapure water is added to bring the reactor's effective working volume to a certain level. Biochar and nano-ferric oxide are then added, and a weak magnetic field is applied. The specific method for applying the weak magnetic field is as follows: A permanent magnet is fixed to the bottom of the reactor, and the magnetic field strength is controlled by adjusting the magnet spacing; a teslameter is used to measure the magnetic field strength. The reactor is continuously stirred until the cumulative gas production increase is less than 1% for three consecutive days, at which point the operation is stopped and the reaction ends.

[0008] Furthermore, the reactor equipped with a single-valve gas collection bag has an effective working volume of 200 mL.

[0009] Furthermore, the amount of silage waste added is 30 g / 200 mL, and the amount of sludge added is 5 g VSS / L.

[0010] Furthermore, the biochar addition amount is 10 g / L.

[0011] Furthermore, the amount of nano-ferric oxide added is 4 g / L.

[0012] Furthermore, the total suspended solids content of the sludge is 25%, and volatile suspended solids account for 68.5% of the total suspended solids wet weight.

[0013] Furthermore, the weak magnetic field specifically refers to: The magnetic field strength at the center point of the reactor is 17.5 ± 0.2 mT.

[0014] Furthermore, the reactor is continuously stirred at 110 r / min at 37±1°C.

[0015] Furthermore, the nano-iron oxide has a purity of 99.5% and a particle size of 15-20 nm.

[0016] Furthermore, the silage waste is stored in sealed plastic bags and quickly transferred to a laboratory refrigerator for refrigeration at 4°C for later use.

[0017] The beneficial effects of this invention are as follows: This invention discloses a method for synergistically enhancing the anaerobic digestion of silage waste using biochar and nano-magnetite driven by a weak magnetic field. This method, through the combined application of a weak magnetic field, nano-ferric oxide, and biochar, increases methane production by 67.7% during the anaerobic digestion of silage waste, enhances acetic acid production, and accelerates the conversion of acetic acid to methane. Under the drive of the weak magnetic field, the conductive network formed by biochar and nano-ferric oxide synergistically reduces the charge transfer impedance of the system, enhances the Fe(III) / Fe(II) redox cycle and interspecies direct electron transfer, and simultaneously increases the activity of key enzymes such as acetate kinase. This promotes the formation of a tighter metabolic interaction network between bacteria and archaea, ultimately achieving efficient coupling of carbon, electron, and energy flows within the anaerobic digestion system. This provides a promising technical strategy for improving the resource utilization efficiency of organic waste. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. The drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 The curves showing the change of soluble chemical oxygen demand (SCOD) with anaerobic digestion time for each treatment group in this invention are shown. Figure 2 The curves showing the change of total ammonia nitrogen (TAN) with anaerobic digestion time for each treatment group in this invention are shown. Figure 3 This is a comparison chart of the total solids (TS) removal rate, volatile solids (VS) removal rate, and SCOD removal rate at the end of digestion for each treatment group in this invention; Figure 4 This is a graph showing the change in the composition of volatile fatty acids (VFAs) over time in each treatment group of the present invention (acetic acid, propionic acid, butyric acid, valeric acid, and isobutyric acid). Figure 5 The curves showing the change of acetic acid concentration with anaerobic digestion time in each treatment group of this invention are shown. Figure 6 The curves showing the change of propionic acid concentration with anaerobic digestion time in each treatment group of this invention are shown. Figure 7 The curves showing the pH change of each treatment group system with anaerobic digestion time are shown below. Figure 8 The curves showing the daily methane production of each treatment group in this invention as a function of anaerobic digestion time are shown. Figure 9 The curves showing the cumulative methane production of each treatment group in this invention as a function of anaerobic digestion time are shown. Figure 10 This is a comparison of cyclic voltammetry (CV) curves of anaerobic sludge samples from different treatment groups in this invention. Figure 11 This is a comparison of the Nyquist plots obtained by fitting the electrochemical impedance spectroscopy (EIS) of each treatment group in this invention; Figure 12 The curves showing the change of Fe(II) concentration in each treatment group of this invention with anaerobic digestion time are shown. Figure 13 The curves showing the change of Fe(III) concentration in each treatment group of this invention with anaerobic digestion time are shown. Figure 14 This is a schematic diagram of the Fe(III) / Fe(II) redox cycle during the anaerobic digestion process of this invention; Figure 15 Radar chart showing the relative activities of key functional enzymes (cellulase, acetate kinase, F420 coenzyme, and cytochrome c) in each treatment group of this invention; Figure 16 This is a correlation analysis diagram showing the relationship between electron transport rate and the activity of key functional enzymes in this invention; Figure 17 This is a diagram showing the composition of bacterial communities at the phylum level in each treatment group on day 2 of this invention. Figure 18 This is a diagram showing the composition of bacterial communities at the phylum level in each treatment group on day 14 of this invention. Figure 19 This is a diagram showing the composition of bacterial communities at the phylum level in each treatment group on day 32 of this invention. Figure 20 This is a diagram showing the composition of archaeal communities at the genus level in each treatment group on day 2 of this invention; Figure 21 This is a diagram showing the composition of archaeal communities at the genus level in each treatment group on day 14 of this invention. Figure 22 This is a diagram showing the composition of archaeal communities at the genus level in each treatment group on day 32 of this invention. Figure 23 This is a diagram of the bacterial-archaic co-occurrence network on day 2 of this invention; Figure 24This is a diagram of the bacterial-archaic co-occurrence network on day 14 of this invention; Figure 25 This is a diagram of the bacterial-archaic co-occurrence network on day 32 of this invention; Figure 26 This is a diagram of the co-occurrence network of bacteria and archaea throughout the entire life cycle under magnetic field-free conditions according to the present invention. Figure 27 This is a diagram of the full-cycle bacterial-archaic co-occurrence network under weak magnetic field conditions according to the present invention. Figure 28 This is a comparison chart of the number of nodes and the number of edges in the co-occurrence network at different stages of the present invention; Figure 29 This is a comparison chart of the average weighting at different stages of the present invention; Figure 30 This is a comparison chart of the modular coefficients of the co-occurrence network at different stages of this invention; Figure 31 This is a comparison chart of the average path length of co-occurring networks at different stages of the present invention; Figure 32 This is a comparison diagram of the network diameters of co-occurring networks at different stages of the present invention; Figure 33 This is a comparison chart of the number of network connected components under no magnetic field and weak magnetic field conditions according to the present invention; Figure 34 This is a comparison chart of the average path length under no magnetic field and weak magnetic field conditions of the present invention; Figure 35 This is a comparison diagram of the network diameter under no magnetic field and weak magnetic field conditions of the present invention; Figure 36 This is a comparison diagram of other key topological parameters under no magnetic field and weak magnetic field conditions of the present invention; Figure 37 This is a comparative chart showing the predicted intensity of effects or functional responses of key metabolic pathways (such as glycolysis, acetyl-CoA generation, propionic acid / butyric acid metabolism, methanogenesis pathway, etc.) based on KEGG in this invention. Figure 38 This is a Sangye diagram of anaerobic digestion according to the present invention; Figure 39 This invention utilizes a weak magnetic field, biochar, and nano-ferric oxide to synergistically construct a conductive network, and enhances Fe... 2+ / Fe 3 + A schematic diagram illustrating the mechanism by which redox cycles promote direct interspecies electron transfer (DIET), thereby enhancing the activity of key enzymes and methane production. Figure 40 The fitted curves and residual analysis diagrams of the methane generation kinetics of the CK treatment group based on the modified Gompertz equation are shown in this invention. Figure 41The fitted curves and residual analysis diagrams of the methane generation kinetics of the CS treatment group based on the modified Gompertz equation are shown in this invention. Figure 42 The fitted curves and residual analysis diagrams of the methane generation kinetics of the CF treatment group based on the modified Gompertz equation are shown in this invention. Figure 43 The fitted curves and residual analysis diagrams of the methane generation kinetics of the CE-treated group based on the modified Gompertz equation are shown in this invention. Figure 44 The figures show the fitting curves and residual analysis diagrams of the methane generation kinetics of the MK treatment group based on the modified Gompertz equation in this invention. Figure 45 The fitted curves and residual analysis diagrams of the methane generation kinetics of the MS treatment group based on the modified Gompertz equation are shown in this invention. Figure 46 The figures show the fitting curves and residual analysis diagrams of the methane generation kinetics of the MF-treated group based on the modified Gompertz equation in this invention. Figure 47 This invention presents the fitting curves and residual analysis diagrams of the methane generation kinetics of the ME treatment group based on the modified Gompertz equation. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0023] In the following embodiments: The silage waste used in this experiment was obtained from Guanling Cattle Germplasm Resource Farm (Guiyang, China), stored in sealed plastic bags, and quickly transferred to a laboratory refrigerator for refrigeration at 4°C. The inoculation sludge was obtained from Zhucheng Haiyam Environmental Protection Technology Co., Ltd. (Shandong, China). The sludge had a total suspended solids (TSS) content of 25%, with volatile suspended solids (VSS) accounting for 68.5% of the total suspended solids (wet weight basis). Biochar was purchased from Changge Longze Purification Agent Co., Ltd. (Henan, China). Nano-ferric oxide (Fe3O4) was analytical grade (99.5% purity), with a particle size of approximately 20 nm, and was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). The permanent magnet was purchased from Minci Technology Co., Ltd. (Shenzhen, China).

[0024] Example 1: Batch anaerobic digestion experiments were conducted in reactors equipped with single-valve gas collection bags (Hebei Jinchang Technology Co., Ltd.), with an effective working volume of 200 mL. 30 g of silage waste was added to each reactor, inoculated with 5 g of VSS / L sludge, and replenished with ultrapure water to a total volume of 200 mL. This study set up eight experimental groups with the following treatments: No magnetic field treatment group: CK (control group), CS (with added biochar), CF (with added nano-ferric oxide), CE (with added biochar + nano-ferric oxide); Magnetic field treatment group: MK (with applied weak magnetic field), MS (with added biochar + applied weak magnetic field), MF (with added nano-ferric oxide + applied weak magnetic field), ME (with added biochar + nano-ferric oxide + applied weak magnetic field). In all treatments, the biochar addition amount was 10 g / L, and the nano-ferric oxide addition amount was 4 g / L. In the weak magnetic field treatment groups, permanent magnets were fixed at the bottom of the reactor, and the magnetic field strength was controlled by adjusting the magnet spacing. The magnetic field strength was measured using a teslameter (KOOT KT-101, China), and the magnetic field strength at the center point of the reactor was 17.5 ± 0.2 mT. The reactor was continuously stirred at 110 r / min at 37 ± 1°C until the cumulative gas production increase was less than 1% for three consecutive days, at which point operation was stopped. Three parallel samples were set up for each experimental condition. Destructive sampling was performed on days 2, 7, 14, 23, and 32, for a total of 8 groups × 5 time points × 3 parallels = 120 reactors. To maintain the anaerobic environment within the reactor, 99.999% pure nitrogen was purged for 3 minutes during sealing.

[0025] Analysis method: Methane production was collected daily using gas collection bags, and the cumulative methane production and daily methane output were calculated. Methane content was determined using a gas chromatograph (Thermo Fisher Scientific Trace 1600, Waltham, USA). Total solids (TS) and volatile solids (VS) content of the samples were determined using standard methods: 10 g of sample was weighed into an aluminum box, dried at 105°C for at least 24 hours to constant weight, and the TTS content was determined; the sample with the TTS determined was then placed in a muffle furnace and incinerated at 550°C for at least 4 hours to constant weight, and the VS content was determined. Soluble chemical oxygen demand (SCOD) was determined using a chemical oxygen demand test kit (Greenkel, China). The pH of the sample suspension was determined using a pH meter (Leici PHSJ-6L, Shanghai, China). Ammonia nitrogen concentration was determined according to the GA Broderick method. Volatile fatty acids (VFAs) were analyzed using a gas chromatograph (Thermo Fisher Scientific Trace 1600, Waltham, USA). The chromatographic system was equipped with an AT-FFAP capillary column (30 m × 0.32 mm × 0.25 µm; Thermo Fisher Scientific, Waltham, USA) and a flame ionization detector (FID). Nitrogen was used as the carrier gas. The constant flow rate was 1.0 mL / min, the split ratio was 40:1, the injection volume was 1 µL, and the injection port and detector temperatures were both set to 250°C. The column temperature program was as follows: initial temperature 90°C, increased to 160°C at 20°C / min, held for 8.5 min, then increased to 170°C at 10°C / min, held for 2 min. Enzyme-linked immunosorbent assay (ELISA) was used, with kits from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (China) to determine the activities of cellulase, acetate kinase, F420 coenzyme, and cytochrome C (CytC) in anaerobic microorganisms. The concentrations of Fe(II) and Fe(III) were determined according to the method of Ferrozin et al. The redox activity and resistivity of anaerobic sludge samples (including solid and liquid fractions) were analyzed using a three-electrode electrochemical workstation (IVIUM n-Stat, Ivium Technologies, Eindhoven, Netherlands) via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. Based on the modified Gompertz equation, a fitting model was used to analyze the methane production of the anaerobic digestion system.

[0026] Microbial community analysis: Microbial DNA was extracted from biomass samples collected on days 2, 14, and 32 using the EZNA® Fecal DNA Kit (Omega Bio-tek, Norcross, USA). The V4-V5 region of the archaea 16S rRNA gene was amplified using primers Arch519F-Arch915R; the V3-V4 region of the bacterial 16S rRNA gene was amplified using primers 341F-806R. The amplified products were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA) according to the manufacturer's instructions. The purified PCR products were quantified using Qubit® 3.0 (Life Invitrogen), and 24 barcoded amplified products were mixed equimolarly. The mixed DNA was then disrupted using an ultrasonic homogenizer, and DNA libraries were constructed using the NEBNext® Ultra™ DNA Library Preparation Kit by denaturing PCR amplification of double-stranded DNA and disruption of non-circularized DNA molecules. Finally, following standard procedures, the library was sequenced using the next-generation sequencing platform (Illumina Miseq) (Shanghai Bio-Tech Co., Ltd.) in PE300 mode.

[0027] Sequences that passed quality control were first deduplicated, then the DADA2 algorithm (the recommended procedure in QIIME 2) was used to identify insertion / deletion mutations (indels) and base substitutions. Paired-end sequences were truncated and filtered based on a maximum expected error of 2 per read (maxEE=2), followed by merging of paired-end sequences and removal of chimeric sequences. Using the uclust algorithm (https: / / github.com / topics / uclust), with an 80% confidence threshold, each 16S rRNA gene sequence (i.e., amplicon variants, ASVs) was aligned to the Silva (SSU138.2) 16S rRNA database (http: / / www.arb-silva.de) for phylogenetic annotation. After generating amplicon variants, 16S rDNA data analysis was performed using the CFViSA platform (http: / / www.cloud.biomicroclass.com / en / CFViSA). PICRUSt2 was used to predict microbial function, focusing on key metabolic pathways related to carbohydrate degradation, methanogenesis, and propionic / butyric acid metabolism. A co-occurrence network was constructed using the WGCNA software package based on 16S rRNA sequencing data.

[0028] Data statistics and analysis methods Data processing and plotting were performed using Origin Pro 2025 software (Origin Lab Corporation, Massachusetts, USA). Each experimental procedure was repeated three times, and the results are presented in the form of averages in the graphs. One-way ANOVA was used to assess statistical significance, and p < 0.05 was considered statistically significant.

[0029] Results and Discussion: Degradation and transformation of organic matter during anaerobic digestion Monitoring the degradation and transformation of organic matter during anaerobic digestion can indirectly assess the digestion efficiency of the system, because these compounds are key substrates for methane formation, such as... Figure 1 As shown, in the early stage of digestion (0-7 days), except for the ME treatment group, the solid organic matter (TS / VS) in all treatment groups gradually decomposed into soluble organic compounds (sugars, oligopeptides, volatile fatty acid precursors, etc.), leading to an increase in the concentration of soluble chemical oxygen demand. Consistent with the results of Zhao Jianwei et al., the rapid hydrolysis process helps microorganisms utilize the fermentation substrate. However, under weak magnetic field conditions, the synergistic effect of biochar and nano-ferric oxide in the ME treatment group shortened the hydrolysis acidification stage, achieved rapid conversion of organic matter, increased the acetic acid ratio, and accelerated propionic acid degradation. Figure 5 , Figure 6 This indicates that ME treatment not only promotes the conversion of recalcitrant organic matter into usable intermediates during the hydrolysis and acidification stage, but also enhances the simultaneous consumption of soluble substrates during the subsequent methanogenesis stage, improving the synchronicity of hydrolysis and methanogenesis processes and reducing the risk of intermediate metabolite accumulation in the system. At the end of digestion, the ME-treated group achieved the highest soluble chemical oxygen demand removal rate, reaching 81.64%. Figure 3 The total ammonia nitrogen (TAN) concentration in all treatment groups increased initially. Figure 2 This is mainly attributed to the decomposition of organic nitrogen compounds (such as proteins). During anaerobic digestion, excessively high total ammonia nitrogen concentrations increase the proportion of free ammonia, exerting toxic effects on methanogenic archaea, ultimately leading to an imbalance in the anaerobic microbial community structure and even system collapse. In this experiment, the total ammonia nitrogen concentration in the anaerobic digestion system did not reach the inhibition range of 1500-7000 mg / L described by Heinfeld et al. (2009). However, the synergistic effect of weak magnetic field, biochar, and nano-ferric oxide reduced the total ammonia nitrogen concentration in the ME treatment group by an average of 38.02% compared to the CK treatment group throughout the anaerobic digestion process. At the end of anaerobic digestion, the total solids removal rate and volatile solids removal rate of the CE, MS, MF, and ME treatment groups were significantly higher than those of the CK, CS, CF, and MK treatment groups (p<0.05). Figure 3The higher total solids and volatile solids removal rates indicate that under ME treatment conditions, organic matter is degraded more thoroughly during anaerobic digestion and is more easily converted into biogas. This is consistent with the higher methane production in this group. Figure 9 This method is consistent with the principle of reducing the volume and active ingredients of residual solid waste, thereby reducing the risk of environmental pollution.

[0030] Volatile fatty acids are key intermediate products in the conversion of complex organic matter into methane during anaerobic digestion, and their production and consumption indirectly reflect the methane production efficiency of the anaerobic digestion system. Figures 4-6 The study showed the relative concentration changes of volatile fatty acids such as acetic acid and propionic acid during anaerobic digestion. Due to the near-complete decomposition of butyric acid, valeric acid, and isobutyric acid, the relative concentration of acetic acid gradually increased. In terms of absolute concentration, the acetic acid levels in the ME and CE treatment groups were significantly higher than those in other treatment groups on days 2 and 7 (p<0.001); at the end of digestion, the ME treatment group had the highest average relative acetic acid concentration (44.5%). Importantly, the absolute conversion of acetic acid in the ME treatment group was approximately 4.2 times that of the CK treatment group. Acetic acid is a major methane precursor (accounting for approximately 72% of methane production) and a key substrate for acetic acid-producing methanogens, ensuring not only efficient energy conversion but also maintaining the dynamic balance between acidification and methanogenesis. The ME and CE treatments significantly increased acetic acid production, promoting the rapid accumulation of complex organic matter into acetic acid-type intermediates while ensuring timely and efficient conversion of acetic acid into methane. This indicates that these two treatments, to some extent, enhanced the acetic acid-type methanogenesis pathway, avoiding excessive accumulation of acetic acid and the risk of acidification within the system. Propionic acid is difficult to degrade and tends to accumulate during anaerobic digestion. High concentrations of propionic acid were observed in all samples on days 2 and 7, with the highest concentrations observed in the MF and ME treatment groups on day 2, reaching 35.07 mmol / L and 37 mmol / L, respectively. The CE, MS, MF, and ME treatment groups exhibited faster propionic acid degradation and conversion rates, indicating that combined application reduced the potential inhibitory effect of organic acid accumulation on the anaerobic digestion process while accelerating the substrate flow towards the methanogenic stage, thereby promoting digestion system stability and increasing methane production. Notably, although the accumulation of volatile fatty acids is typically accompanied by a decrease in pH, the pH of all treatment groups remained weakly alkaline with minimal fluctuations in this experiment. Figure 7 Similar situations were observed in the study by Xu et al., which may be attributed to the ammonia nitrogen released from the inoculated sludge. In addition, the complex composition of the inoculated sludge may have buffered the acidification caused by the increase in the concentration of volatile fatty acids.

[0031] Coupling relationship between methane production and electron transfer Methane, as the final product of anaerobic digestion, is the primary carrier for energy recovery. Based on this, daily methane production and cumulative methane yield curves were plotted. Compared to the control group, under both natural and weak magnetic field conditions, all treatment groups showed increased methane yield in the initial stage of anaerobic digestion (days 5-8), with the ME treatment group exhibiting the highest methane yield on day 5 (1.96 ml / g). Figure 8 This indicates that the combined application of a weak magnetic field, biochar, and nano-ferric oxide promotes the rapid activation of acid-producing and methanogenic bacteria. Firstly, both biochar and nano-ferric oxide are excellent carriers and conductive pathways, enhancing microbial attachment and electron transfer efficiency. Secondly, the weak magnetic field may increase cell membrane permeability and enzyme activity, accelerating the flow of energy from the substrate to the methanogenic stage, allowing the system to enter the high-yield stage more quickly. Subsequently, the methane yield growth in the ME and CS treatment groups was disrupted, possibly because the recalcitrant inhibitors released during anaerobic digestion inhibited anaerobic microorganisms and methanogenic activity, thus reducing methane production. In fact, methanogenic bacteria are more sensitive to environmental fluctuations than hydrolytic or acid-producing bacteria; if methanogenic bacteria adapt to the environment and conditions improve, yield will show a "secondary rebound," consistent with previous findings. After 16 days of anaerobic digestion, daily methane production decreased sharply, and after 25 days, cumulative methane production tended to stabilize. Figure 9 The ME treatment group had the highest cumulative methane production, reaching 14.82 ml / g (p<0.05), followed by the CE treatment group (12.6 ml / g), representing increases of 67.7% and 42.5% respectively compared to the CK treatment group. This is likely due to the three treatments collectively constructing a more efficient interspecies direct electron transport network, improving the utilization efficiency of electron donors by methanogenic archaea, thereby achieving a higher methane conversion rate. Table 1 shows that the maximum cumulative gas production (Pm) of the combined treatment groups (CE, MS, MF, ME) was significantly increased, with the ME treatment group reaching 14.45 mL / g VS, a 66% increase compared to the CK treatment group; the lag period (λ) of the combined treatment groups was approximately 5.4–5.9 days, shortened by 0.7–1.2 days. This indicates that the combined treatments promoted the rapid initiation of the methanogenesis process and increased the microbial initiation rate. This may be because biochar and nano-iron oxide provided attachment sites for microorganisms, while the weak magnetic field increased cellular metabolic activity, enabling the methanogenic community to establish a stable metabolic network more quickly. The regression model shows that all treatment groups have good stability and reliability, such as Figures 40-47 The ME, MS, and MF treatment groups showed higher residual dispersion and more complex kinetic changes in the methane generation process. This indirectly indicates that applying a weak magnetic field and adding biochar and nano-Fe3O4 effectively altered the metabolic balance and gas production performance of the anaerobic digestion system.

[0032] Table 1 Gas dynamics fitting parameters ; Nano-ferric oxide and biochar established close interactions with anaerobic microorganisms during the process of mediating direct interspecies electron transfer, thereby regulating the redox activity and conductivity of the synthetic microbial community. Compared with the control group (CK), under weak magnetic field conditions, the cyclic voltammetric curves of the MS, MF, or CE treatment groups showed obvious reduction and redox peaks. Figure 10 The increased redox peak current indicates a significant improvement in the conversion rate of oxidized and reduced substances in the extracellular electron transport chain of microorganisms, and enhanced electron carrier turnover efficiency. This is beneficial for methanogens to efficiently utilize metabolites (such as hydrogen and acetic acid) and inhibit the accumulation of intermediate metabolites. The ME, MF, and CE treatment groups all exhibited excellent performance, demonstrating the reversible Fe3O3 of nano-Fe3O3. 2+ / Fe 3+ Redox cycles can serve as efficient electronic mediators, accelerating extracellular electron transfer processes. Furthermore, the excellent conductivity and magnetism of nano-Fe3O4 contribute to the formation of a more continuous microbial conductive network, thereby enhancing direct interspecies electron transfer between acid-producing and methanogenic bacteria.

[0033] Electrochemical impedance spectroscopy can characterize the impedance characteristics of biological cathodes under different electrical stimulation modes. The fitted Nyquist plot is shown below. Figure 11 As shown in the figure, the results revealed significant differences in impedance among the treatment groups. The ME treatment group exhibited the smallest radius of curvature, corresponding to the lowest charge transfer resistance and the highest electron transport efficiency, consistent with the significant increase in methane production in this group. The reduced impedance indicates an effective enhancement of the electron transport pathway between acid-producing and methanogenic bacteria, forming a more continuous conductive channel within the system. This not only lowers the hydrogen partial pressure in the system, promoting metabolic coupling between propionic / butyric acid oxidizing bacteria and hydrogen-nutritive methanogenic bacteria, but may also enhance the acetate-nutritive methanogenesis pathway. This explains the dual advantages of the ME treatment group in terms of both methane yield and cumulative methane production.

[0034] Figure 14 This study demonstrates the Fe(III) / Fe(II) redox cycle in anaerobic digestion, a pathway for electron acceptance and supply that enables electron transfer and recycling, crucial for accelerating organic matter degradation and methane production. Figure 12 As shown, during the methanogenesis stage, Fe(II) is gradually released as iron oxide is reduced during methane formation, peaking around day 14 and then gradually decreasing. The CF-treated group showed a relatively high Fe(II) dissolution concentration, indicating a higher degree of reduction by nano-Fe3O4; while the MF and ME-treated groups showed lower Fe(II) dissolution concentrations than the CF-treated group, suggesting that the constant magnetic field may accelerate the Fe(III) / Fe(II) redox cycle (i.e., promote Fe...). 2+(Re-oxidation or consumption), thereby reducing the accumulation of Fe(II) in the liquid phase. The Fe(III) dissolution concentration in the ME-treated group was also consistent with its rapid turnover characteristics ( Figure 13 This improved turnover efficiency may be attributed to the fact that the weak magnetic field increased the magnetization of the nano-Fe3O4, thereby increasing its electron mobility and surface reactivity, and thus enhancing the electron flux during the iron cycling process. However, the Fe(III) dissolution concentration in the MF-treated group was significantly higher than that in the CE-treated group. This may be because the reducing surface functional groups (such as -OH) in the CE-treated group can reduce Fe(III) to Fe(II), thereby inhibiting Fe in the liquid phase. 3+ The accumulation of Fe(III) / Fe(II) was observed. Although both the MF and ME treatment groups were affected by weak magnetic fields, the combination of biochar and ME may have shifted the Fe(III) / Fe(II) cycle to a solid-phase process. Through the coupling of adsorption-complexation and high-flux interspecies direct electron transfer, the accumulation of Fe(III) / Fe(II) in the liquid phase was significantly reduced. After the lag period, the Fe(II) concentration began to decrease, indicating that the Fe(II) generated in the early reduction stage was subsequently re-oxidized. At the same time, the Fe(III) concentration continued to increase, further confirming the continuous occurrence of the iron redox cycle within the system. This finding provides new insights and technical references for optimizing anaerobic digestion systems, mitigating environmental pollution, and improving the efficiency of renewable energy recovery.

[0035] like Figure 15 As shown in the radar chart, the relative activities of cellulase, acetate kinase, F420 coenzyme, and cytochrome c compared to the CK treatment group were depicted. The ME treatment group showed higher activities of all four target enzymes, with F420 coenzyme and cytochrome c activities increasing by approximately 51.45% and 27%, respectively, compared to the CK treatment group (p<0.05). During anaerobic digestion, F420 coenzyme primarily acts as an intracellular electron carrier in methanogenic archaea, participating in the key step of mediating CO2 reduction to CH4; while cytochrome c participates in extracellular or transmembrane electron transport, serving as a crucial medium for direct interspecies electron transport, especially when combined with conductive materials, significantly enhancing electron transport efficiency. Therefore, in the ME treatment group, the synergistic effect of the weak magnetic field and biochar / nano-iron oxide significantly improved the efficiency of direct interspecies electron transport, promoting effective metabolic coupling between acid-producing bacteria and methanogenic archaea, accelerating organic matter degradation, and thus increasing methane production. While other treatment groups showed some promoting effects, the enhancement was limited.

[0036] The ME-treated group exhibited the highest acetate kinase activity. Acetase, a key enzyme in acetic acid production metabolism, enables acid-producing bacteria to generate acetic acid, which is a direct substrate for methanogenesis. Notably, the ME-treated group also showed the highest acetic acid concentration, consistent with the aforementioned results. Correlation analysis was performed on electron transport intensity and the activity of key metabolic enzymes (…). Figure 16The results showed that electron transfer intensity was positively correlated with the activity of all tested enzymes, and some correlations reached statistical significance (p<0.05), confirming that enhanced electron transfer in the system was closely related to the upregulation of key metabolic enzyme activity.

[0037] Furthermore, strong correlations exist between different enzyme systems, with the coupling effect between cellulase and F420 coenzyme / cytochrome c being the most significant, indicating a significant synergistic relationship between substrate degradation, electron transport, and energy metabolism within the anaerobic digestion system. These findings further demonstrate that, under the combined effects of a weak magnetic field, nano-iron oxide, and biochar, the anaerobic digestion system forms a synergistic metabolic network of "rapid substrate hydrolysis - efficient electron transport - enhanced methane production." This network promotes the simultaneous improvement of energy conversion efficiency at multiple metabolic levels, which is the fundamental mechanism behind the highest methane production in the ME treatment group.

[0038] Microbial community structure analysis Previous studies have shown that adding conductive materials alters the structure of functional microorganisms in anaerobic digestion systems, thereby affecting organic matter degradation efficiency, electron transfer processes, and is directly related to methanogenesis performance. Figures 17-19 The changes in bacterial communities at the phylum level during different stages of anaerobic digestion were shown. Firmicutes (Bacillota), Bacteroidetes, Pseudomonadota, and Chloroflexota were the dominant phyla in the fermentation process, playing crucial roles in hydrolysis and acidification. The relative abundance of Firmicutes was highest in the CF treatment group (30.70%), followed by the MF treatment group (28.23%), maintaining a high level on days 2 and 14 before declining later. This is because Firmicutes, as Gram-positive bacteria, prefer readily degradable carbon sources and are essential for crude protein degradation and sugar reduction in anaerobic digestion. In the later stages of anaerobic digestion, soluble sugars and starch are largely consumed, and the system shifts from hydrolysis-driven acidification to a process dominated by acetic acid and methane production.

[0039] Bacteroides can degrade a variety of organic compounds and is positively correlated with volatile fatty acid production. Although the relative abundance of Firmicutes and Bacteroides was higher in the ME treatment group, the Bacteroides / Firmite ratio remained stable at all stages (average approximately 0.93, close to 1.0). In contrast, this ratio decreased significantly in the CK and CE treatment groups (CK: 0.83→0.73; CE: 1.01→0.74), while the ratios in the MS and MF treatment groups were both >1.0, which may indicate that Bacteroides are over-dominant, and the acidification process is out of balance with the acetic acid production process. These results indicate that the ME treatment achieves dynamic functional coupling between acidification and acetic acid production, avoids the accumulation of intermediate products such as propionic acid, ensures a continuous supply of acetic acid, and thus promotes efficient methanogenesis. This is consistent with the excellent gas production potential and rate of the ME treatment group in the Gompertz kinetic analysis (Table 1).

[0040] The abundance of Proteobacteria decreased in most treatment groups, but the application of a weak magnetic field generally increased its proportion. Proteobacteria include electroactive bacteria capable of extracellular electron transfer (e.g., direct interspecies electron transfer between the electrode and the microorganism). Notably, although the ME treatment group had the lowest relative abundance of Proteobacteria, this group exhibited the best electrochemical performance. Figures 10-11 Due to the diverse metabolic functions of Proteobacteria, some bacteria compete with methanogens for electron acceptors, and their reduced abundance can decrease the diversion of electrons to non-methanogenic pathways. Therefore, despite the low abundance of Proteobacteria, ME treatment optimized the microbial community structure and enhanced direct interspecies electron transfer under the promotion of iron cycling, thereby achieving the highest substrate removal rate, electrochemical activity, and methane production.

[0041] Figures 20-22 The temporal changes in archaeal communities at the genus level were shown. On day 2, all treatment groups were dominated by *Methanothermobacter* (a typical hydrogen-trophic methanogen, utilizing H2 / CO2 to produce methan). Its significant enrichment under non-magnetic conditions indicated that the hydrogen-trophic methanogenesis pathway was dominant at this point, while the significant decrease under magnetic conditions suggested that the magnetic field enhanced direct interspecies electron transfer, inhibited the traditional H2-mediated electron transfer pathway, and altered the dominant methanogenic community structure. On day 14, the relative abundance of *Methanothermobacter* decreased to 14.2-38.5%, while the abundance of *Candidatus nitrosopumilus* increased. This shift reflects the transition of anaerobic digestion from an early rapid methanogenesis phase to a phase where multifunctional metabolic processes coexist. At this stage, enhanced ammonia oxidation and nitrogen cycling begin to compete with methanogenesis, reducing the dominance of methanogens, consistent with the observed decrease in methane yield at this stage. Figure 8Under weak magnetic field conditions, the abundance of SCGC AAA011-D5 increased significantly, but its functional role in anaerobic digestion remains unclear due to a lack of direct evidence. Notably, *Methanocorpusculum* (another hydrogen-trophic methanogen) was more abundant in the non-magnetic field treatment group, indicating that methanogenesis relies on conventional H2-mediated interspecies electron transport rather than direct interspecies electron transport in the absence of enhanced magnetic field, suggesting that the introduction of a magnetic field may inhibit the H2 pathway by increasing conductivity and electron transport efficiency. By day 32, the anaerobic digestion system had reached maturity, with a stable microbial community structure and an overall decrease in the abundance of thermoautotrophic methanogens. Importantly, the ME treatment group maintained high levels of *Nitrostrophobic Dwarf* (15.17%) and thermoautotrophic methanogens (22.85%) on day 32, demonstrating a synergistic effect of enhanced nitrogen cycling and stable methanogenesis.

[0042] Evolution and magnetic field regulation of bacterial-archaic co-occurrence networks at different stages To elucidate the dynamic evolution of microbial interactions during anaerobic digestion, a co-occurrence network of bacteria and archaea at the phylum level was constructed at days 2, 14, and 32. Figures 23-25 By comparing topology and network parameters ( Figures 28-32 This study reveals the evolutionary trend of microbial interactions from initial dispersion to later synergy.

[0043] On day 2, the microbial interaction network was relatively sparse, highly modular, and had low connectivity, consistent with the characteristics of the anaerobic initiation phase. At this time, complex organic matter was the main substrate, and bacterial communities (such as Firmicutes) dominated the hydrolysis and acidification processes. Cooperative interactions related to methanogenesis had not yet formed a clear network. On day 14, the network became significantly denser, with an increase in the number of nodes and connections, and enhanced positive correlations, indicating a stronger metabolic interdependence between bacteria and archaea. Volatile fatty acid degradation products were utilized by intermediate metabolic bacteria. The increase in topological parameters confirmed that the system had entered an intermediate state of metabolic synergy and stable symbiosis.

[0044] On day 32, the network reached its most complex and integrated state, forming a highly interconnected network dominated by dense positive correlations, with peak microbial co-metabolism and energy flow. An efficient direct / indirect electron transport network (interspecific direct electron transport / interspecific indirect electron transport) was established between Firmicutes (hydrolysis), Chlorobacteria (hydrogen / acetic acid production), and Archaea (methanogenesis). Temporal changes in network parameters (significantly reduced connected components, increased network diameter, average path length, and average weighting; p < 0.05) indicate that microbial interactions gradually shifted from dispersed, weak interactions to close cooperation and efficient information transfer. This evolution from a dispersed to a highly connected cooperative network provides a structural basis for the rapid transfer and simultaneous transformation of intermediate metabolites (soluble chemical oxygen demand, volatile fatty acids, H2, and acetic acid) during organic matter degradation. It facilitates the sequential utilization by acid-producing and methanogenic bacteria, avoids the accumulation of intermediate products, and lays the foundation for improved soluble chemical oxygen demand removal rates, stable volatile fatty acid conversion, and increased methane production through microbial interactions.

[0045] To investigate the ecological interactions between bacteria and archaea under magnetic field conditions, a full-cycle co-occurrence network was constructed for both the non-magnetic field and magnetic field treatment groups. Notably, the network structures differed significantly: the non-magnetic field network ( Figure 26 The fewer node connections and higher modularity of the magnetic field network indicate that the ecological niches are dispersed and coexist, and the metabolic synergy is weak; while the magnetic field network ( Figure 27 The bacteria are significantly denser, with increased number and thickness of connections, improved connectivity, and reduced modularity, indicating enhanced complexity of interactions and metabolic coordination between bacteria and archaea.

[0046] Increased connectivity between Firmicutes / Green Curvatures and Broad Archaea indicates that the magnetic field accelerates energy coupling between hydrogen-producing / acetogenic bacteria and methanogens by enhancing direct interspecies electron transfer. This high-density bacterial-archaeal interaction network facilitates the oxidation of reducing volatile fatty acids (propionic and butyric acids), bypassing the traditional H2-mediated limiting steps and reducing hydrogen partial pressure inhibition during propionic acid oxidation. This also explains the rapid degradation of propionic acid, accelerated accumulation of acetic acid, and efficient methanogenesis in the ME and CE treatment groups. The magnetic field induced significant changes in topological parameters (connectivity components, average path length, network diameter); Figures 33-36 (p<0.05) enhances the overall network complexity and connectivity, strengthens the metabolic cascade reaction among hydrolytic bacteria, acid-producing bacteria and methanogenic bacteria, and enables the conversion of solid organic matter into soluble intermediate products (soluble chemical oxygen demand) to proceed synchronously with the subsequent methanogenic process.

[0047] Metabolic pathway prediction and potential mechanism of weak magnetic field-biochar-nano iron oxide promoting anaerobic digestion of silage waste To systematically reveal the response patterns of carbon flux conversion and energy metabolism under different conditions, a comparative analysis was conducted on key reaction nodes in major metabolic stages (such as carbohydrate hydrolysis and glycolysis, acetyl-CoA synthesis, acidification, and methanogenesis) based on the metabolic pathway map of the Kyoto Encyclopedia of Genetics and Genomes (KEGG). Figure 37 ).

[0048] During the carbohydrate hydrolysis and glycolysis stages, the conversion of cellulose and starch into glycolytic intermediates (such as glucose-6-phosphate and pyruvate) was activated in most treatment groups, indicating that the addition of substrate and conductive material alone can enhance the initial decomposition and digestion activity of organic matter. The synergistic effect of weak magnetic field, biochar, and nano-Fe3O4 significantly upregulated the expression of genes encoding multiple enzymes involved in propionic acid degradation, including genes encoding propionyl-CoA synthase, methylmalonyl-CoA epimerase, and methylmalonate semialdehyde dehydrogenase.

[0049] In the CO2 reduction-methanation pathway, both the application of a weak magnetic field alone and its synergistic effect with biochar / nano-iron oxide induced aldehyde dehydrogenase (NAD). + The expression of genes encoding formylmethanefuran dehydrogenase, N5-formyl-tetrahydromethanepterin transferase, 5,10-methylene-tetrahydromethanepterin cyclase, 5,10-methylene-tetrahydromethanepterin dehydrogenase, and 5,10-methylene-tetrahydromethanepterin reductase was increased, simultaneously enhancing the F420 coenzyme-dependent redox reaction. As a key intracellular electron carrier in hydrogen-dependent methanogenesis, the increased activity of F420 coenzyme indicates that weak magnetic fields, biochar, and nano-ferric oxide significantly enhanced intracellular reducing power supply. This may be attributed to improved extracellular interspecies direct electron transport, promoting electron flux into methanogenic archaea and driving a synchronous upregulation of the overall flux of the CO2 reduction pathway.

[0050] During the pyruvate dehydrogenase-catalyzed reaction and acetyl-CoA synthesis stages, the ME-treated group exhibited significant metabolic activation, with an effect strength generally reaching 1.0. This indicates that the synergistic effect of these factors significantly promoted substrate oxidation and electron release, accelerating the conversion of carbon flux from carbohydrate intermediates to acetyl-CoA (a core energy metabolism substrate), thereby improving the overall metabolic rate and energy transfer efficiency of the system. As a central metabolic hub connecting carbohydrate metabolism, fatty acid metabolism, and the methanogenesis pathway, the significant increase in acetyl-CoA production flux signifies the simultaneous activation of multiple downstream functional modules, improving system-level electron release efficiency and substrate recovery rate.

[0051] During the methanogenesis stage, the synergistic effect of the three significantly enhanced the reduction efficiency of the terminal electron acceptor (CO2 or acetic acid), with the related pathway effect strength reaching 1.0, indicating a significant improvement in the metabolic activity of the methanogenic archaea and the system's electron supply capacity. Notably, the ME-treated group reached peak activity in both the propionic acid metabolic pathway and the methanogenesis pathway (a highly reducing metabolic pathway), suggesting that the synergistic application of the weak magnetic field, biochar, and nano-ferric oxide lowered the electron transfer energy barrier, enabling electrons to migrate along low-energy pathways and improving the system's electron utilization efficiency.

[0052] To further elucidate the microbial symbiotic mechanism and electron transport pattern behind this metabolic enhancement effect, a Sankey diagram was constructed representing the relationship between "treatment conditions → functional microbial community → electron transport type → functional enzyme activity → methane production". Figure 38 The synergistic effect of weak magnetic field, biochar, and nano-ferric oxide significantly altered the dominance of the main functional microbial community and the distribution of electron transport pathways, thereby regulating the activity of key metabolic enzymes and ultimately affecting methane production capacity. The ME treatment group formed a stable interspecies direct electron transport network, enhanced the activity of key enzymes (cellulase, acetate kinase (AK), and F420 coenzyme) and methane yield, and achieved efficient carbon flux conversion and maximum energy release.

[0053] The combined application of weak magnetic fields, biochar, and nano-ferric oxide significantly improved the anaerobic digestion performance of silage waste. For example... Figure 39 As shown, biochar and nano-ferric oxide form a conductive network, establishing a stable electronic pathway between hydrolyzing bacteria and methanogenic bacteria, reducing dependence on the diffuse intermediate H2, thereby improving coupling efficiency. Their synergistic effect not only strengthens the Fe... 2+ / Fe 3+ Redox cycles promote direct interspecies electron transfer and improve volatile fatty acid conversion efficiency and methane production capacity by stabilizing pH. A weak magnetic field enhances the conductivity of the nano-ferric oxide-biochar composite system, improving electron transfer efficiency while simultaneously promoting microbial metabolism, upregulating enzyme activity (such as acetate kinase and F420 coenzyme), and stabilizing pH and metabolic conditions by regulating the system's redox homeostasis. Existing research indicates that improvements in anaerobic digestion performance stem from enhanced microbial activity, rather than alterations in community structure.

[0054] Overall, the combined application of weak magnetic fields, biochar, and nano-Fe3O4 enhances electron transport efficiency by activating the iron redox cycle and interspecies direct electron transport, synergistically boosting the activity of key metabolic enzymes, and reconstructing a stable interaction network centered on "hydrolytic acid-producing bacteria - highly efficient, low-abundance methanogenic archaea." From a system integration perspective, the ME treatment group constructs a highly coupled carbon flux-electron flux-energy flux synergistic metabolic network through "network reconstruction at the structural level - enhanced electron transport at the process level - synergistic activation of enzyme systems at the functional level," which is also the fundamental mechanism for achieving maximum methane production and the fastest kinetic response.

[0055] In this experiment, the combined application of a weak magnetic field (WMF), nano-ferric oxide (NF), and biochar (BC) increased methane production by 67.7% in the anaerobic digestion of silage waste, enhanced acetic acid production, and accelerated the conversion of acetic acid to methane. Driven by the weak magnetic field, the conductive network formed by biochar and nano-ferric oxide synergistically reduced the charge transfer impedance of the system, strengthened the Fe(III) / Fe(II) redox cycle and interspecies direct electron transport (DIET), and simultaneously increased the activity of key enzymes such as acetate kinase and F420 coenzyme. This promoted the formation of a tighter metabolic interaction network between bacteria and archaea, ultimately achieving efficient coupling of carbon, electron, and energy flows within the anaerobic digestion system. In conclusion, this study demonstrates that the combined application of a weak magnetic field, biochar, and nano-ferric oxide significantly optimizes the anaerobic digestion performance of silage waste, providing a promising technical strategy for improving the resource utilization efficiency of organic waste.

[0056] Therefore, those skilled in the art will recognize that although embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Therefore, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.

Claims

1. A method for synergistically enhancing the anaerobic digestion of silage waste using a weak magnetic field-driven biochar and nano-magnetite, characterized in that, The method is as follows: In a reactor equipped with a single-valve gas collection bag, silage waste is added, sludge is inoculated, and ultrapure water is added to bring the reactor's effective working volume to a certain level. Biochar and nano-ferric oxide are then added, and a weak magnetic field is applied. The specific method for applying the weak magnetic field is as follows: The permanent magnet is fixed at the bottom of the reactor, and the magnetic field strength is controlled by adjusting the spacing between the magnets; Magnetic field strength was measured using a teslameter. The reactor is continuously stirred until the cumulative gas production increase is less than 1% for three consecutive days, at which point the operation is stopped and the reaction ends.

2. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The reactor equipped with a single-valve gas collection bag has an effective working volume of 200 mL.

3. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The amount of silage waste added is 30 g / 200 mL, and the amount of sludge added is 5 gVSS / L.

4. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The biochar addition amount is 10 g / L.

5. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The amount of nano-ferric oxide added is 4 g / L.

6. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The total suspended solids content of the sludge is 25%, and volatile suspended solids account for 68.5% of the total suspended solids wet weight.

7. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The weak magnetic field is specifically defined as follows: the magnetic field strength at the center point of the reactor is 17.5 ± 0.2 mT.

8. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The reactor was continuously stirred at 110 rad / min at 37±1°C.

9. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The nano-ferric oxide has a purity of 99.5% and a particle size of 15-20 nm.

10. The method for synergistic enhancement of anaerobic digestion of silage waste by weak magnetic field-driven biochar and nano-magnetite as described in claim 1, characterized in that, The silage waste was stored in sealed plastic bags and quickly transferred to a laboratory refrigerator for refrigeration at 4°C for later use.

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