Continuous Anaerobic Digestion Research Articles

Maximizing biogas production from leftover injera: influence of yeast addition to anaerobic digestion system

Abstract Injera is a staple food in Ethiopian dine. This study aimed to investigate on leftover injera (LI) for producing biogas via anaerobic digestion (AD), while leftover injera is full of easily biodegradable components. Aiming to examine the impact of yeast addition on biogas production efficiency, it was found that the addition of 2 % volatile solids (VS) of stimulated yeast, daily biogas output increased by 520 and 550 ml after 12 and 37 days of anaerobic digestion, respectively, with rather steady biogas production. The rate at which gas production increased was drastically cut in half when yeast was left out of the control group. Biogas production increased by only 60 ml despite the addition of two portions of substrate and yeast. Biogas output in the yeast group after fermentation was also up 33.2 % compared to the control group. The yeast group’s anaerobic digesting system was more stable, as determined by the study of markers including volatile organic acids, alkalinity, and propionic acid. The findings can be used as a benchmark for future trials aiming to industrialise continuous anaerobic digestion, allowing for more flexible response to feed as waste LI as organic load.

Towards a sustainable energy future: Evaluating Arundo donax L. in continuous anaerobic digestion for biogas production

AbstractIn response to the EU's REPowerEU initiative (COM (2022) 108) which encourages an increase in biogas production by 20% in member states by 2030 to boost energy independence, it has become essential to identify sustainable alternatives to traditional feedstocks for biogas production, especially in the EU Member states where there is still high dependence on corn silage as the main raw material in biogas plants. While corn silage, predominantly used in the European biogas plants today, serves primarily for the livestock sector, alternative sources need to be explored. Therefore, this study aimed to evaluate the potential of Arundo donax, a perennial energy crop, as an alternative feedstock in a continuous anaerobic process. The biogas yield and its quality, characterized by CH4, CO2, H2S and O2 content, were determined during a continuous process with A. donax, compared with two mixed feedstocks of A. donax and corn silage over a 5‐month period in a continuous anaerobic digestion process. The results revealed that A. donax exhibits a biogas yield and methane content comparable to corn silage, indicating its potential as a viable and sustainable alternative feedstock for biogas production.

A hybrid approach of anaerobic digestion model no. 1 and machine learning to model and optimize continuous anaerobic digestion processes

Anaerobic digestion is a promising approach to dispose of biodegradable waste and wastewater, generating biogas as an alternative energy resource. This work proposed a so-called M-CADM1 for continuous anaerobic digestion simulation, which combined the machine learning and anaerobic digestion model No.1 (ADM1). The detailed reaction path and intermediate products in different stages of anaerobic digestion are specified in ADM1. The kinetic parameters were modified by machine learning. The characteristics (elemental composition) of feedstocks were used to predict kinetic parameters. A total of 75 biomass samples were used to establish for machine learning models. Five element contents (C, H, O, N, S), feedstock feed rate, and anaerobic digestion temperature were used as the input. The kinetic parameters were set as output. The sensitivities of 17 kinetic parameters were evaluated. 7 kinetic parameters with the highest sensitivities were selected as ADM1 model inputs by sensitivity analysis. The R2 and RMSE were used as the index to evaluated the accuracy of machine learning model. The best R2 and RMSE reached 0.84 and 0.196. The TIC was used as the index to evaluated the accuracy of M-CADM1. By comparing the simulated value with the experimental value, the accuracy of the overall M-CADM1 expressed by TIC of kitchen waste was 0.036. The organic acid content and pH in the reactor were considered as indicators to study the accuracy and stability of the M-CADM1. Trends in organic acids, free ammonia or hydrogen inhibition, and pH were consistent with experimental continuous anaerobic digestion results.

Effect of iron powder on hydrogen sulfide reduction and phosphate removal in semi‐continuous anaerobic digestion treating primary sludge

AbstractBACKGROUNDTo our best knowledge, no experimental study has previously investigated the simultaneous effects of adding Fe powder for the decrease of hydrogen sulfide (H2S) in biogas and the removal of phosphate (PO4‐P) in continuous anaerobic digestion (AD). This study investigated the effects of iron (Fe) powder (5 g L−1) in AD to remove H2S and PO4‐P. A comparative experiment was conducted using two semi‐continuous‐flow anaerobic digesters treating primary sludge at decreasing hydraulic retention times (HRTs) of 20 to 10 days.RESULTSThe addition of Fe powder to AD removed > 98% of H2S at all HRTs, and removed 64–72% of PO4‐P, with the highest removal efficiency at HRT = 10 d. The addition of Fe powder to AD had no noticeable effect on biogas production or pH, but it increased the CH4 content (62.7–66.0% in Fe‐treated vs 58.2–60.4% in the Control) partly due to the greater presence of Methanoregulaceae. The relative abundance of the microbial groups was more affected by HRT than by the addition of Fe powder. The presence of Fe powder notably increased the abundance of Spirochaetaceae and Syntrophaceae.CONCLUSIONAs shown, the addition of Fe powder can improve the management of both H2S in the biogas and PO4‐P in the digested sludge in the AD system. © 2024 Society of Chemical Industry (SCI).

Extraction of bioethanol from fermentation broth and valorization of the vinasse for biogas production

An effective ethanol extraction from fermentation broths and further utilization of the waste streams for biogas production can enhance the overall efficiency of the biorefinery process. In the present study, ethanol was extracted from a fermentation broth by liquid-liquid extraction (LLE) after ultrafiltration. Thereafter, the vinasse was subjected to batch and continuous anaerobic digestion (AD). Batch AD was carried out at inoculum-to-substrate ratios (ISR) of 0.1, 0.2 and 0.3 (gVS/gCOD), while continuous AD was carried out at hydraulic retention times (HRT) of 2, 3 and 5 days as well as at organic loading rates (OLR) of 2, 5 and 7 gCOD/L/day. Results showed that during ultrafiltration, fouling mechanism was primarily due to the formation of a cake layer on the surface of the membrane. LLE efficiency was 97.13% with an ethanol purity of 98.84%. During the batch AD, the highest cumulative biomethane potential of 236.43 mL/gCOD (± 13) was obtained at an ISR of 0.2 (gVS/gCOD). During the continuous AD, both OLR and HRT significantly affected biogas and biomethane production with the highest biogas yield being 415.50 mL/gCOD at an HRT of 5 days and OLR of 2 gCOD/L/day.

Effect of different organic loads on the performance and microbial community mechanism of dry anaerobic digestion

In this study, the vertical continuous push-flow dry anaerobic digestion (AD) reactor was developed independently. Typical agricultural organic waste straw and cow dung were used as raw materials in this paper to carry out long-term continuous mixed dry AD experiments on a controlled laboratory scale. The experimental results showed that the system was in low-stress operation at an organic load rate (OLR) of 3 g VS L-1 d-1, the VS methane production rate was located at 225 ∼ 272 mL CH4 g VS-1, and the volumetric methane production rate was located at 0.95 ± 0.08 L (L·d)-1, and the combination of the volumetric methane production rate, VS methane production rate, methane content of the gas, and the VS removal rate showed that the system's operational performance in this stage was in the best condition in the whole set of experiments. When the OLR reached 25 g VS L-1 d-1, the content of VFAs all began to climb rapidly above 8000 mg/L, with the content of acetic acid increasing most significantly due to the fact that the activity of acetic acid-type methanogenic bacteria was the first to be inhibited. Eventually, because of the kinetic differences between the hydrolytic acidifying bacteria and the methanogenic archaea, the final acidification destabilisation of the anaerobic digestion reactor occurred and the system was irreversibly destabilised. The system performance at this stage was in the best condition in the whole set of experiments. The study of microbial community succession and metabolic mechanisms showed that Methanoculleus and Methanobacterium play a key role in sustaining methane production, and the increased abundance and dominance of hydrogen methanogens implies that methane is produced via the hydrogen methanogenic pathway under high organic loading conditions.

Determination of operational parameters for the first stage of continuous temperature-phased anaerobic digestion of oily food waste: Influent concentration, hydraulic retention time, pH control and temperature

Anaerobic digestion is promising in both food waste reduction and energy upgrading. However, the intermediates in anaerobic digestion of such an oily food waste, long chain fatty acids, not only have low bioavailability but also significantly threaten operation stability. Temperature-phased anaerobic digestion is an emerging technology in recent years, expected to overcome those issues. Given significance of the first stage of the two-stage process, continuous thermophilic digestion of oily food waste was conducted, with mesophilic digestion operated in parallel as control, to determine practical operational parameters from operation performance and stability. The feeding concentration at approximately 4.50 % of volatile solids was identified to be applicative for a relatively stable operation. Due to slow β-oxidation for long chain fatty acids, hydraulic retention time for the first stage needed longer than 10 d to maintain anaerobes’ survival so that they were not inhibited. In addition to the risk from inhibition, readily acidified risk still existed, and instant pH control was demonstrated to be efficient in maintaining the habitat of microbial metabolism. Compared with no viable bacteria detected under uncontrolled pH, 36.7 % live cells could still be detected under controlled pH. Overall, thermophilic digestion obviously achieved advantages in higher substance degradation and methane production with lower stability risk. The genera Methanosarcina and Methanothermobacter played a significant role, with the ratio at about 2:1. While bacterial viability was not reduced remarkably in preliminary period of inhibition, long-term operation under high lipid loading resulted in its loss.

Investigation into the effects of different recycled magnetic additives on anaerobic co-digestion of sludge and straw

The addition of magnetic additives has been found to enhance the anaerobic co-digestion of sludge and straw. Due to strong magnetic properties, these additives can be easily separated from the digestate, allowing for recycling during the continuous anaerobic digestion process. This study aimed to investigate the impact of different recycled magnetic additives (specifically, magnetic biochar and Fe3O4) on the anaerobic co-digestion of sewage sludge with corn straw. The results showed that the addition of recycled Fe3O4 with an initial amount of 10 g/kg resulted in the highest cumulative methane yield of 295.70 mL/g VSadded, which increased by 79.81% as compared to the control. The results suggest that magnetic biochar with higher carbon content exerts a more noticeable impact on shortening the digestion lag phase time, as indicated by kinetic analysis. With the addition of recycled Fe3O4, the Methanosaeta and Methanosarcina enhanced significantly. Especially Methanosarcina, which was increased from 3.85% to 6.28%, resulting in a 2.43% increase. Furthermore, the predominant aceticlastic pathway of digestion process was consolidated, and the enriched Methanosarcina broadened the pathways of methanogenesis. This research provides a potential utilization strategy of recycling Fe3O4 for accelerating the anaerobic degradation of urban and rural areas solid waste.

Generation of High Concentration Free Volatile Fatty Acids from Continuous Anaerobic Digestion of Chicken Manure by Suppressing the Decomposition of Nitrogenous Components.

Free volatile fatty acids (free VFA) play a crucial role in the inactivation of pathogens during the anaerobic digestion of animal manure. However, the decomposition of nitrogenous components can release alkaline ammonium-N, which might increase the pH and reduce the concentration of free VFA. In this study, continuous anaerobic digestion of high-solid chicken manure was conducted for 150days. The results indicated the process stabilized at a pH of approximately 6.0, with total ammonia nitrogen (TAN) of around 7.0g/L. The resulting concentration of free VFA was only about 3.1g/L, which might not sufficiently effective for pathogen inactivation. On the 70th day, hydrogen chloride was added into the reactor to adjust the pH to 5.5. This led to a further decrease in pH to 4.3 and TAN to 2.3g/L. As a result, the concentration of free VFA significantly increased, reaching up to 12.6g/L. These findings support the potential for generating high levels of free VFA even for nitrogen-rich manure by implementing an appropriate process regulation.

Effect of removal of inhibitors on microbial communities and biogas yield of Jatropha curcas seeds during continuous anaerobic digestion

Jatropha curcas seeds, as an abundant lignocellulosic biomass, offer a highly promising and ideal alternative for producing energy in the form of methane. Use of J. curcas seeds has the potential to significantly bolster the biofuel sector, fostering a more sustainable circular economy. In the current study, different fractions of processed J. curcas seeds were investigated for biogas production. J. curcas seed pressed cake, a by-product of biodiesel production, was subjected to methanolic extraction. The remaining solids, referred to as methanolic residues, yielded more biogas in batch experiments than pressed cake and residues from aqueous and n-hexane extractions. The compounds extracted with methanol inhibited hydrolysis and reduced biogas production by 35.5% compared to the same setup without extracts. In continuous reactors fed with methanolic residues, the highest biogas yield occurred at an organic loading rate (OLR) of 1 g VS L−1 day−1 and a hydraulic retention time (HRT) of 20 days. The relative abundance of acetogenic bacteria was higher in reactors fed with methanolic residues than in those fed with seed pressed cake, seed oil, and whole seed. Jatropha seed oil and whole seed did not inhibit methanogens. A higher relative abundance of methanogenic communities was observed in all reactors at HRT of 20 days compared to those at HRTs at 15 and 10 days. These findings can be used to increase biogas production during anaerobic digestion of J. curcas seed components and suggests a zero-waste biorefinery production route for value added compounds derived from the removal of biogas-inhibiting components.

Anaerobic Digestion of Olive Mill Wastewater and Process Derivatives—Biomethane Potential, Operation of a Continuous Fixed Bed Digester, and Germination Index

Olive mill wastewater (OMW) management is an economic and environmental challenge for olive oil-producing countries. The recovery of components with high added value, such as antioxidants, is a highly researched approach that could help refinance performant wastewater treatment systems. Anaerobic (co-)digestion is a suitable process to valorize the energetic and nutritional content of OMW and OMW-derived waste streams from resource recovery processes. Issues of process stability, operation, and yields discourage industrial application. Deepening the understanding of biomethane potential, continuous anaerobic digester operational parameters, and co-substrates is key to large-scale implementation. The biomethane potential of different OMW-derived samples and organic solid market waste as co-substrate was 106–350 NL methane per kg volatile solids (VS). The highest yields were obtained with the co-substrate and depolyphenolized OMW mixed with retentate from an ultrafiltration pretreatment. Over 150 days, an anaerobic fixed-bed 300 L digester was operated with different OMW-derived substrates, including OMW with selectively reduced polyphenol concentrations. Different combinations of organic loading rate and hydraulic retention time were set. The biogas yields ranged from 0.97 to 0.99 L of biogas per g of volatile solids (VS) eliminated, with an average methane content in the produced biogas of 64%. Potential inhibition of the process due to high polyphenol concentrations or over-acidification through volatile fatty acids was avoided in the continuous process through process and substrate manipulation. High concentrations of potassium and low concentrations of nitrogen and phosphate end up in the digestate. Sulfate reduction results in high H2S concentrations in the biogas. The digestate was tested for phytotoxic properties via the germination index. Diluted digestate samples improved germination by up to 50%.

Anaerobic co-digestion of expired alcohol-based hand sanitizer with synthetic wastewater for biogas production

In this work, anaerobic co-digestion was demonstrated as a viable method for utilizing​ expired alcohol-based hand sanitizer from COVID-19 response for additional biogas production. Co-digestion experiments were conducted using three parallel continuous flow anaerobic digesters. The results highlight the importance of acclimatisation to avoid process instability. Process instability was observed when co-digesting ethanol-based sanitizer (at 0.3% v/v) with sewage sludge without acclimatisation. However, the digester was fully recoverable and a gradual increase in the addition of ethanol-based sanitizer showed stable and good performance even at 0.6% v/v. The specific biogas production was in the range of 295–304 mL/gCOD.day and the COD removal efficiency was 76%. When isopropanol-based sanitizer was used as a co-substrate, digester previously acclimatised with ethanol-based sanitizer showed 20% higher biogas production than another digester that has not been acclimatised. These results highlight the need for an acclimatisation period so that the microbial community can adjust to the new substrate. Furthermore, due to the easily degradable nature of alcohol-based sanitizer and synthetic wastewater in this study, there was a threshold of organic loading rate of 3.5–4 gCOD/L.day. This threshold can be explained by the accumulation of volatile fatty acids that could inhibit the methanogenesis process. Results in this study demonstrate anaerobic co-digestion as a sustainable option for valorizing expired alcohol-based hand sanitizer.

Using biochemical methane potential results for the economic optimization of continuous anaerobic digestion systems: the effect of substrates’ synergy

During the processing of raw agricultural materials from the primary sector, large quantities of by-products with high chemical energy content are produced. Some of these are olive mill wastewater (OMW), cheese whey (CW), and liquid cow manure (LCM). Anaerobic digestion is a promising, environmentally friendly process for the energy valorization of agro-industrial and livestock by-products. The purpose of this study was the mathematical modeling of anaerobic digestion in batch systems, aiming to quantify possible synergies occurring between complementary substrates. The analysis was based on using biochemical methane potential data to predict the optimum hydraulic retention time (HRT) under steady-state conditions of continuous systems, for different substrate ratios. The objective function for HRT optimization was based on maximizing the process profit and included technoeconomic parameters, allowing the comparison of the different substrate ratios that can be used in the feedstock. The co-digestion with a mixture ratio of OMW:LCM equal to 70:30 proved very satisfactory for the sustainability of the process, exhibiting a maximum net profit of 16 €/m3reactor/d for a plant capacity of 10 m3feed/d.

Determination residence time distribution of the solid phase in a dry anaerobic semi-continuous horizontal reactor of 0.5 m3 using innovative 3D printed tracers

Solid residence time distribution (SRTD) analysis is important in the design, modeling, and operation of solid biomass in continuous dry anaerobic digestion (D-AD) reactors. SRTD analysis could allow the appropriately adjust of the applicable organic charge and the study of unusable volume due to solid accumulation and preferential pathways. The lack of sustainable, environmentally friendly, and cost-effective solid tracers limits the study of solid flow patterns in D-AD digesters. In this study, 3D printing enabled the fabrication of customized Poly Lactic Acid (PLA) tracers with a specific shape to perform SRTD analysis of the semi-continuous digestion of straw-cattle manure (SCM) in a horizontal reactor of 0.5 m3. The recovery of tracers at the reactor outlet allowed the determination of the average SRTD at 29.43 days. Solid convection was predominant over diffusion, indicating that the reactor behaves closer to a plug flow reactor (PFR) rather than a complete stirred tank reactor.

Enhanced hydrogen and methane production from date fruit wastes using semi continuous two-stage anaerobic digestion process with increasing organic loading rates

Biological conversion of date wastes under continuous conditions is a challenge due to their high content of biodegradable organic matter. In this study, hydrogen (H2) and methane (CH4) production in a two-stage process to increase the energy recovery from date pulp wastes (DPW) was investigated. In the first stage, the effect of increasing organic leading rate (OLR) on dark fermentation (DF) performances was evaluated in a semi continuously stirred tank reactors (SCSTR) without nutrients addition. The highest volumetric H2 production rate (VHPR) and specific H2 yield (SHY) of 0.76 L/L.d and 81 LH2/kg volatile solid (VS), respectively were achieved at high OLR of 11.74 gVS/L.d. In the second stage, the DF effluent was used as substrate to feed the SCSTR of anaerobic digestion (AD). The highest volumetric methane production rate (VMPR) of 2.92 L/L.d was achieved with a high OLR (4.25 gVS/L.d) and short hydraulic retention time (HRT, 8 days), producing specific methane yield (SMY) of 530 LCH4/kgVS. Thus, the two-stage process can be an effective strategy for high rate renewable energy recovery (13 kJ/gVSinlet) and organic waste processing.

Combined sodium citrate and ultrasonic pretreatment of waste activated sludge for cost effective production of biogas

This study aimed to pretreat the waste activated sludge (WAS) by ultrasonication in an energy efficient way by combining sodium citrate with ultrasonic pretreatment at 0.03 g/g suspended solids (SS) of dosage. The ultrasonic pretreatment was done at various (20–200 W) power levels, sludge concentration (7 to 30 g/L), sodium citrate dosages (0.01 to 0.2 g/g SS). An elevated COD solubilization of 26.07 ± 0.6 % was achieved by combined pretreatment at a treatment time of 10 min, ultrasonic power level of 160 W when compared to individual ultrasonic pretreatment (18.6 ± 0.5 %). A higher biomethane yield of 0.26 ± 0.009 L/g COD was achieved in sodium citrate combined ultrasonic pretreatment (SCUP) than ultrasonic pretreatment (UP) 0.145 ± 0.006 L/g COD. Almost 50% of the energy can be saved through SCUP when compared to UP. Future study evaluating SCUP in continuous mode anaerobic digestion is vital.

Excess methane production and operation stability for anaerobic digestion of oily food waste controlled by mixing intensity: Focusing on heterogeneity of long chain fatty acids

Long chain fatty acids (LCFAs) are the key intermediate of anaerobic digestion of oily food waste, not completely soluble in a water-dominant anaerobic system due to their long hydrocarbon chains with hydrophobic property. Their effective concentration affects release of high methanogenic potential and system stability. A long-term continuous anaerobic digestion of oily food waste demonstrated excess methane production of even more than feedstock in an anaerobic continuous stirred tank reactor (CSTR). Assuming feedstock COD at 100%, approximately 120% of COD as methane could be achieved. Oil floating and crystallization with Ca salt resulting from the distribution heterogeneity of LCFAs in the CSTR were found responsible for the excess methane production. Moreover, slow conversion and accumulation of saturated LCFAs with relatively lower solubility played an important role as well. Compared with unsaturated oleic (C18:1) and linoleic acids (C18:2), around twice slower methane production rate and longer lag time could be observed for those saturated LCFAs. Mixing intensity was proved to be a critical controlling factor for methanogenesis and stability possibly by affecting interaction between oil/LCFAs and anaerobes to change effective lipid loading.

Biogas Production from Residues of Industrial Insect Protein Production from Black Soldier Fly Larvae Hermetia illucens (L.): An Evaluation of Different Insect Frass Samples

Insect biomass shows promise as an alternative animal feedstuff with a low climate effect. Industrial insect rearing generates residual materials, such as feed remains and insect excrements, so-called insect frass, which exhibits a high organic content. Commonly, these residues are utilized as soil amendment. Information on the suitability of these residues for biogas production is rather scarce. The energetic utilization of insect frass as feedstock for anaerobic digestion (AD) would allow for the simultaneous residue material reduction and bioenergy production. Additionally, synergies in heat management could arise using the exhaust heat of the biogas plant in the insect farming process. In laboratory-scale anaerobic digestion trials, the specific methane yield (SMY) of six different insect frass samples from black soldier fly (Hermetia) rearing were tested in batch biochemical methane potential (BMP) tests. Further, semi continuous anaerobic digestion trials on a lab scale using continuously stirred tank reactors (CSTRs) were carried out with Hermetia insect frass from a pilot plant operation in order to determine the digestibility and process stability of the AD process. The BMP results showed SMY values of the different insect frass samples ranging from 201 ± 9 to 287 ± 37 mL/gVS that are similar to those of other animal excrements, such as cow or pig manure already been used as feedstock in agricultural biogas plants. Results of the semi-continuous digestion of insect frass from the pilot plant operation showed a SMY value of 167 ± 15 mL/gVS, suggesting no process-inhibiting effect caused by the feed material. Although, the high nitrogen content must be taken into account for stable AD performance.

The Role of Slurry Reflux in a Corn Stalk Continuous Anaerobic Digestion System: Performance and Microbial Community.

Slurry reflux is a low-cost slurry reduction technology, which can solve the problem that a large amount of slurry cannot be completely consumed in a biogas plant. Anaerobic digestion (AD) of corn stalks with slurry reflux and non-reflux was compared and evaluated in continuous anaerobic digestion to clarify the effects of slurry reflux on AD with organic loading rate (OLR) variation. It was found that slurry reflux increased cumulative methane production and improved system stability. The average methane yield of the slurry reflux group was 224.19 mL/gVS, which was 41.35% higher than that of the non-reflux group. High-throughput sequencing results showed that slurry reflux increased the microbial community richness. The dominant microorganisms in the reflux group were in phylum Bacteroidetes, which have the capacity to degrade polymers, and Methanothrix, which is an aceticlastic methanogen. The relative abundances of Bacteroidetes and Methanothrix were 32.41% and 41.75%, respectively. Clostridium III and Saccharofermentans, which are related to syntrophic acetate oxidation and hydrolysis, were increased in relative abundance in the slurry reflux system. The increase of the OLR altered the main methane-producing pathway from the acetoclastic methanogenic pathway to the hydrogenotrophic methanogenic pathway in the AD system, and the slurry reflux can delay this trend. This study provided an effective way for the reduction and utilization of slurry in a biogas plant.

Biochar regulates enzymes activity and interspecies electron transfer to promote bioenergy recovery from a continuous two-stage food waste anaerobic digestion process

The influence of biochar on the performance of food waste anaerobic digestion in a continuous two-stage process was examined. Biochar was added to a methanogenic (MG) reactor, with 50% of its effluent recirculated to a hydrolytic–acidogenic (HA) reactor. The addition of biochar in the MG reactor simultaneously enhanced the performance of both reactors, and the energy recovery efficiency increased by 25.01–70.99%. Biochar improved the activities of protease and α-amylase in the HA and MG reactors by 1.08–2.23 and 0.46–1.21, and 13.65–15.76 and 8.73–13.64 times, respectively. The enhancement of these hydrolytic enzymes' activity in the HA reactor was correlated to the effluent recirculation from the MG reactor, contributing to the improved hydrolysis of food waste. Furthermore, the activity of coenzyme F420 in the MG reactor increased by 11.47%, 18.81%, and 27.03%, respectively, indicating that biochar can promote hydrogenotrophic methanogenesis. Microbial analysis revealed that the presence of biochar promoted interspecies hydrogen transfer between Syntrophomonas and hydrogenotrophic methanogens. Additionally, biochar triggered direct interspecies electron transfer between Geobacter sulfurreducens and acetotrophic methanogens, enabling effective methanogenesis.