Enhanced methane production from waste activated sludge by potassium ferrate combined with thermal hydrolysis pretreatment.

Anaerobic digestion of waste activated sludge (WAS) was one of the directions of sludge treatment, but how to effectively improve the production of methane as a resource product of anaerobic digestion of sludge still needs further research. The study examined how the combination of potassium ferrate (PF) and thermal hydrolysis (TH) pretreatment affected methane production from sludge. The results demonstrated a positive synergistic effect on methane production with PF-TH pretreatment. Specifically, by employing a 0.05 g/g TSS (total suspended solids) PF in conjunction with TH at 80 °C for 30 min, the methane yield increased from 170.66 ± 0.92 to 232.73 ± 2.21 mL/g VSS (volatile suspended solids). The co-pretreatment of PF and TH has been substantiated by mechanism studies to effectively enhance the disintegration and biodegradability of sludge. Additionally, the variation of microbial community revealed an enrichment of active microorganisms associated with anaerobic digestion after treated with PF + TH, resulting in a total abundance increase from 11.87 to 20.45% in the PF + TH group.

Enhanced degradation and methane production of food waste anaerobic digestate using an integrated system of anaerobic digestion and microbial electrolysis cells for long-term operation.

The integrated system of anaerobic digestion and microbial electrolysis cells (AD-MEC) was a novel approach to enhance the degradation of food waste anaerobic digestate and recover methane. Through long-term operation, the start-up method, organic loading, and methane production mechanism of the digestate have been investigated. At an organic loading rate of 4000 mg/L, AD-MEC increased methane production by 3-4 times and soluble chemical oxygen demand (SCOD) removal by 20.3% compared with anaerobic digestion (AD). The abundance of bacteria Fastidiosipila and Geobacter, which participated in the acid degradation and direct electron transfer in the AD-MEC, increased dramatically compared to that in the AD. The dominant methanogenic archaea in the AD-MEC and AD were Methanobacterium (44.4-56.3%) and Methanocalculus (70.05%), respectively. Geobacter and Methanobacterium were dominant in the AD-MEC by direct electron transfer of organic matter into synthetic methane intermediates. AD-MEC showed a perfect SCOD removal efficiency of the digestate, while methane as clean energy was obtained. Therefore, AD-MEC was a promising technology for deep energy transformation from digestate.

Food waste impact on dry anaerobic digestion of straw in a novel reactor: Biogas yield, stability, and hydrolysis-methanogenesis processes.

Gradient anaerobic digestion reactor (GADR) can improve substrate utilization efficiency by solving the problem of the "short circuit" of materials. However, the substrate's composition significantly affects the reactor's performance. This study investigated the impact of food waste (FW) levels on corn straw's dry anaerobic digestion (AD) in a novel GADR. The results show that biomethane production can be improved by coupling urban and agricultural solid waste recycling. The mechanism is to increase the hydrolysis and acid production efficiency, and the abundance of enzymes related to methanogenesis. The maximum methane yield (494.2 mL CH4/g VS) and the highest anaerobic biodegradability (85.7 %) were obtained when the FW was added at 60 %. The co-digestion of FW and straw can improve the hydrolysis and acid production efficiency and methane yield, which improves the buffering capacity and stability of the system compared with the single digestion of FW.

Ensemble machine learning prediction of anaerobic co-digestion of manure and thermally pretreated harvest residues.

This study aimed to clarify the statistical accuracy assessment approaches used in recent biogas prediction studies using state-of-the-art ensemble machine learning approach according to 10-fold cross-validation in 100 repetitions. Three thermally pretreated harvest residue types (maize stover, sunflower stalk and soybean straw) and manure were anaerobically co-digested, measuring biogas and methane yield alongside eight thermal preprocessing and biomass covariates. These were the inputs to an ensemble machine learning approach for biogas and methane yield prediction, employing three feature selection approaches. The Support Vector Machine prediction with the Recursive Feature Elimination resulted in the highest prediction accuracy, achieving the coefficient of determination of 0.820 and 0.823 for biogas and methane yield prediction, respectively. This study demonstrated an extreme dependency of prediction accuracy to input dataset properties, which could only be mitigated with ensemble machine learning and strongly suggested that the split-sample approach, often used in previous studies, should be avoided.

A robust, low-temperature, closed-loop anaerobic system for high-solid mixed farm wastes: advancing agricultural waste management solutions in Canada.

This study investigates the effectiveness of low-temperature (20 ± 1 °C) anaerobic digestion (AD) for two organic multiple farm substrate combinations: Set 1 comprising chicken manure (CM), dairy manure (DM), and waste corn silage (CS) and Set 2 comprising CM, DM, pig manure (PM), and CS. Inoculum adaptation steps were carried out using CM and CM+DM for Set 1 and Set 2, respectively. Over three consecutive operating cycles spanning 245 days with increasing organic loads, 4.3 and 2.8 g VS L-1 d-1 for Sets 1 and 2 during Cycles 1 to 5.1 and 4.6 g VS L-1 d-1for Sets 1 and 2 during Cycle 3, a closed-loop two-stage liquid-solid AD system was employed, with performance assessed via stability ratios of short-chain volatile fatty acids and alkalinity. Results demonstrate that mono-digestion of CM with adapted inoculum yielded the highest biogas production of 424 ± 4 L over 77 days, indicating superior performance by Set 1 during Phase I, whereas a similar performance was observed during Phase 2, where Sets 1 and 2 exhibited highest specific methane yields of 0.233 ± 0.028 and 0.262 ± 0.004 L g-1 VSfed, respectively, over 68 days. Analysis of heavy metal concentrations in digestates revealed a significant decrease compared to initial raw substrate concentrations, highlighting their role as nutrients for microbial growth. This study, the first of its kind, highlights the potential of low-temperature AD systems to manage diverse organic residues/byproducts and offers insights into effective performance monitoring without compromising system integrity.

A data-driven approach for revealing the linkages between differences in electrochemical properties of biochar during anaerobic digestion using automated machine learning.

Biochar is commonly used to enhance the anaerobic digestion of organic waste solids and wastewater, due to its electrochemical properties, which intensify the electron transfer of microorganisms attached to its large surface area. However, it is difficult to create biochar with both high conductivity and high capacitance, which makes selecting the right biochar for engineering applications challenging. To address this issue, two Auto algorithms (TPOT and H2O) were applied to model the effects of different biochar properties on anaerobic digestion processes. The results showed that the gradient boosting machine had the highest predictive accuracy (R2 = 0.96). Feature importance analysis showed that feedstock concentration, digestion time, capacitance, and conductivity of biochar were the main factors affecting methane yield. According to the two-dimensional (2D) partial dependence plots, high-capacitance biochar (0.27-0.29 V·mA) is favorable for substrates with low-solid content (< 19.6 TS%), while the high-conductivity biochar (80.82-170.58 mS/cm) is suitable for high-solids substrates (> 20.1 TS%). The software, based on the optimal model, can be used to obtain the ideal range of biochar for AD trials, aiding researchers in practical applications prior to implementation.

Potential mechanisms of propionate degradation and methanogenesis in anaerobic digestion coupled with microbial electrolysis cell system: Importance of biocathode.

Microbial electrolysis cells (MEC) have the potential for enhancing the efficiency of anaerobic digestion (AD). In this study, microbiological and metabolic pathways in the biocathode of anaerobic digestion coupled with microbial electrolysis cells system (AD-MEC) were revealed to separate bioanode. The biocathode efficiently degraded 90 % propionate within 48 h, leading to a methane production rate of 3222 mL·m-2·d-1. The protein and heme-rich cathodic biofilm enhanced redox capacity and facilitated interspecies electron transfer. Key acid-degrading bacteria, including Dechloromonas agitata, Ignavibacteriales bacterium UTCHB2, and Syntrophobacter fumaroxidans, along with functional proteins such as cytochrome c and e-pili, established mutualistic relationships with Methanothrix soehngenii. This synergy facilitated a multi-pathway metabolic process that converted acetate and CO2 into methane. The study sheds light on the intricate microbial dynamics within the biocathode, suggesting promising prospects for the scalable integration of AD-MEC and its potential in sustainable energy production.

Biowaste management by hydrothermal carbonization and anaerobic co-digestion: Synergistic effects and comparative metagenomic analysis.

The feasibility of anaerobic co-digestion in semicontinuous mode of two major urban biowaste, food waste (FW) and garden and park waste (GPW) (75 % FW and 25 % GPW) as well as the co-digestion of FW with the process water originated from the hydrothermal carbonization of GPW (95 % FW and 5 % process water), both on a COD basis, has been assessed. The effect of varying organic loading rate (OLR) from 1.5 to 3.5 g COD/L·d on methane yield, gross energy recovery, and microbiome population was evaluated. For comparison, anaerobic digestion of FW was also conducted to determine the best strategy for sustainable biowaste management. This study showed an optimal OLR of 2.5 g COD/L·d. Acetic and propionic acid content increased as OLR raised for each condition studied, while methane yield decreased at the highest OLR tested indicating overloading of the system. The anaerobic co-digestion of FW and process water showed a 10 % increase on methane production compared to anaerobic digestion of FW (324 vs. 294 mL CH4 STP/L·d). Moreover, it enhances the process due to a greater abundance and diversity of hydrolytic and acidogenic bacteria belonging to Bacterioidota, Firmicutes, and Chloroflexi phyla, as well as promotes the hydrogenotrophic pathway under higher propionic concentrations which is not usually favoured for methane production. The integration of hydrothermal carbonization of GPW with the anaerobic co-digestion of 95 % FW and 5 % of process water results in the highest potential energy recovery and could be a good strategy for sustainable management of urban biowaste.

Anaerobic digestion of wastewater from hydrothermal liquefaction of sewage sludge and combined wheat straw-manure.

Hydrothermal liquefaction (HTL) shows promise for converting wet biomass waste into biofuel, but the resulting high-strength process water (PW) requires treatment. This study explored enhancing energy recovery by anaerobic digestion using semi-batch reactors. Co-digesting manure with HTL-PW from wheat straw-manure co-HTL yielded methane (43-49% of the chemical oxygen demand, COD) at concentrations up to 17.8 gCOD·L-1, whereas HTL-PW from sewage sludge yielded methane (43% of the COD) up to only 12.8 gCOD·L-1 and complete inhibition occurred at 17 gCOD·L-1. Microbial community shifts confirmed inhibition of methanogenic archaea, while hydrolytic-fermentative bacteria were resilient. Differences in chemical composition, particularly higher levels of N-containing heterocyclic compounds in PW of sewage sludge, likely caused the microbial inhibition. The considerable potential of combining HTL with anaerobic digestion for enhanced energy recovery from straw-manure in an agricultural context is demonstrated, yet sewage sludge HTL-PW requires more advanced approaches to deal with methanogenesis inhibitors.

Nanobubble water promotes anaerobic digestion of high-solids cattle manure under mesophilic and thermophilic conditions.

The gradual increase in cattle farming has led to a huge production of cattle manure (CM), but the conventional treatment methods are less efficient. In this study, the treatment method of anaerobic digestion (AD) of high-solids CM by combining nanobubble water (NBW) with different gases was proposed to present a new idea for the reduction, harmlessness, and resourcefulness of CM. It was found that the performance of the digester with added NBW was better than the control. Among them, the cumulative methane yield T-Air: 227.09 mL g-1 VSadded and T-CO2: 226.12 mL g-1 VSadded increased by 17.72 % and 17.22 %, respectively, compared with the control T: 192.90 mL g-1 VSadded under thermophilic conditions. Under mesophilic conditions, M-Air: 162.39 mL g-1 VSadded increased by 9.68 % compared with control M: 148.05 mL g-1 VSadded. Microbial communities analyzed at the genus level revealed that the relative abundance of bacteria favorable to hydrolysis and acid-producing processes, such as Defluviitalea, Haloplasma, and Bacillus, increased to varying degrees. Moreover, the relative abundance of archaea favorable for methanogenesis, such as Methanoculleus, Methanobrevibacter, and Methanosarcina, also increased to varying degrees. Therefore, the addition of NBW promoted the hydrolysis of high-solids CM, enhanced the stability of the reaction, improved the methanogenic performance, and increased the RA of favorable genera, which ultimately led to a better performance of the AD of high-solids CM.

Formation/characterization of humin-mediated anaerobic granular sludge and enhanced methanogenic performance.

This study presents a novel method for accelerating the granulation of methanogenic anaerobic granular sludge (AnGS) in an upflow anaerobic sludge blanket (UASB) reactor using solid-phase humin (HM). The results demonstrated that HM-mediated AnGS (HM-AnGS) formed rapidly within 50 days. The increase in particle size, settling velocity and mechanical strength was attributed to the rapid granulation of the HM-AnGS. The maximum methane yield of the HM-AnGS was 5-fold higher than that of the control group. This is consistent with the findings, which showed that HM-AnGS had 3.2-3.4 times more methyl-coenzyme M reductase (Mcr) activity and 2.4-2.9 times more adenosine triphosphate (ATP) than control groups. Molecular analyses indicate that HM most likely accelerated interspecies electron transfer (IET) in HM-AnGS (e.g., from Enterococcus to Methanosaeta). Furthermore, the HM-AnGS was effective in recovering energy from actual slaughterhouse wastewater.

Animal factors that affect enteric methane production measured using the GreenFeed monitoring system in grazing dairy cows.

Similar to all dairy systems internationally, pasture-based dairy systems are under increasing pressure to reduce their greenhouse gas (GHG) emissions. Ireland and New Zealand are 2 countries operating predominantly pasture-based dairy production systems where enteric CH4 contributes 23% and 36% of total national emissions, respectively. Ireland currently has a national commitment to reduce 51% of total GHG emissions by 2030 and 25% from agriculture by 2030, as well as striving to achieve climate neutrality by 2050. New Zealand's national commitment is to reduce 10% of methane emissions by 2030 and between 24% and 47% reduction in methane emissions by 2050. To achieve these reductions, factors that affect enteric methane (CH4) production in a pasture-based system need to be investigated. The objective of this study was to assess the relationship between enteric CH4 and other animal traits (feed intake, metabolic liveweight, energy corrected milk yield, milk urea concentration, and body condition score [BCS]) in a grazing dairy system. Enteric CH4 emissions were measured on 45 late lactation (213.8 ± 29 d after calving) grazing Holstein-Friesian and Holstein-Friesian × Jersey crossbred cows (lactation number 3.01 ± 1.65, 538.64 ± 59.37 kg live weight, and 3.14 ± 0.26 BCS) using GreenFeed monitoring equipment for 10 wk. There was a training period for the cows to use the GreenFeed of 3 wk before the 10-wk study period. The average enteric CH4 produced in the study was 352 g ± 45.7 g per day with an animal to animal coefficient of variation of 13%. Dry matter intake averaged 16.6 kg ± 2.23 kg per day, while milk solids (fat plus protein) averaged 1.62 kg ± 0.29 kg per day. A multiple linear regression model indicated that each one unit increase in energy corrected milk yield, metabolic liveweight and milk urea concentration, resulted in an increase in enteric CH4 production per day by 3.9, 1.74, and 1.38 g, respectively. Although each one unit increase in BCS resulted in a decrease in 39.03 g CH4 produced per day. When combined, these factors explained 47% of the variation in CH4 production, indicating that there is a large proportion of variation not included in the model. The repeatability of the CH4 measurements was 0.66 indicating that cows are relatively consistently exhibiting the same level of CH4 throughout the study. Therefore, enteric CH4 production is suitable for phenotyping.

Performance and mechanism of various microaerobic pretreatments on anaerobic digestion of tobacco straw.

Tobacco straw is an abundant biomass in China's agricultural ecosystems, and has high potential for methane production. However, the anaerobic digestion (AD) efficiency is limited by the recalcitrant lignocellulose structure of the tobacco straw. In this study, three microaerobic pretreatments were performed for the AD of tobacco straw to increase methane production. Among them, microbial pretreatment with biogas slurry at an oxygen concentration of 4 mL/g VS resulted in the highest methane production of 349.1 mL/g VS, increasing by 19.8 % than that of untreated. During this pretreatment, the relative abundances of Enterococcus and Clostridium sensu stricto 12, which are closely related to acetic acid production and cellulose degradation, were high, and these bacteria might have an important contribution to substrate hydrolysis and the methanogenesis efficiency of the AD process. This study advances the understanding of microaerobic pretreatment processes and provides technological guidance for the efficient utilization of tobacco straw.

Machine learning assisted modelling of anaerobic digestion of waste activated sludge coupled with hydrothermal pre-treatment.

This study utilizes decision-tree-based models, including Random Forest, XGBoost, artificial neural networks (ANNs), support vector machine regressors, and K nearest neighbors algorithms, to predict sludge solubilization and methane yield in hydrothermal pretreatment (HTP) coupled with anaerobic digestion (AD) processes. Analyzing two decades of published research, we find that ANN models exhibit superior fitting accuracy for solubilization prediction, while decision-tree models excel in methane yield prediction. Pretreatment temperature is identified as pivotal among various variables, and heating time surprisingly emerges as equally significant as holding time for solubilization and surpasses it for methane yield. Contrary to prior expectations, the HTP method's impact on sludge solubilization and AD performance is minimal. This study underscores data-driven models' potential as resource-efficient tools for optimizing advanced AD processes with HTP. Notably, our research spans nearly two decades of lab, pilot, and full-scale studies, offering novel insights not previously explored.

Utilization of carbon-based nanomaterials for wastewater treatment and biogas enhancement: A state-of-the-art review.

The management of environmental pollution and carbon dioxide (CO2) emissions is a challenge that has spurred increased research interest in determining sustainable alternatives to decrease biowaste. This state-of-the-art review aimed to describe the preparation and utilization of carbon-based nanomaterials (CNM) for biogas enhancement and wastewater contaminant (dyes, color, and dust particles) removal. The novelty of this review is that we elucidated that the performance of CNMs in the anaerobic digestion (AD) varies from one system to another. In addition, this review revealed that increasing the pyrolysis temperature can facilitate the transition from one CNM type to another and outlined the methods that can be used to develop CNMs, including arc discharge, chemical exfoliation, and laser ablation. In addition, this study showed that methane (CH4) yield can be slightly increased (e.g. from 33.6% to 60.89%) depending on certain CNM factors, including its type, concentration, and feedstock. Temperature is a fundamental factor involved in the method and carbon sources used for CNM synthesis. This review determined that graphene oxide is not a good additive for biogas and CH4 yield improvement compared with other types of CNM, such as graphene and carbon nanotubes. The efficacy of CNMs in wastewater treatment depends on the temperature and pH of the solution. Therefore, CNMs are good adsorbents for wastewater contaminant removal and are a promising alternative for CO2 emissions reduction. Further research is necessary to determine the relationship between CNM synthesis and preparation costs while accounting for other factors such as gas flow, feedstock, consumption time, and energy consumption.

Organic stabilization and methane production under different organic loading rates in UASB treating swine wastewater.

This study proposes the was to evaluate the stability and methane production with organic load differents in an upflow anaerobic sludge blanket reactor (UASB) treating swine wastewater by methods of multivariate analysis. Four organic loads were used with average hydraulic holding times of one day. The methods of data analysis of linear regression, Pearson correlation, principal component analysis and hierarchical clustering analysis were used for understanding stability and methane production in the reactor. The highest concentrations of bicarbonate alkalinity of 683 mg L-1 CaCO3 and total volatile acids of 1418 mg L-1 HAc with maximum organic loading applied were obtained. The optimal stability conditions occurred at an intermediate and partial alkalinity ratio between 0.24 and 0.25 observed in initial phases with a chemical oxygen demand (COD) removal of 47-57%. Maximum methane production was 9.0 L CH4 d-1 observed with linear regression positive and occurred at the highest applied organic load, corresponding to the highest COD removal efficiency and increased microbial biomass. Positive and negative correlation between functional stability in anaerobic digestion showed regular activity between acids, alkalinity and organic matter removal. This fact was also proven by the analysis of principal components that showed three components responsible for explaining 83.2% of the data variability, and the alkalinity, organic matter influent and organic acids had the greatest effects on the stability of the UASB reactor. Hierarchical clusters detected the formation of five groupings with a similarity of 50.1%, indicating that temperature and pH were variables with unitary influences on data dimensionality.

Comparative analysis of rumen metagenome, metatranscriptome, fermentation and methane yield in cattle and buffaloes fed on the same diet.

A study to compare the rumen microbial community composition, functional potential of the microbiota, methane (CH4) yield, and rumen fermentation was conducted in adult male cattle and buffaloes fed on the same diet. A total of 41 phyla, 169 orders, 374 families, and 1,376 microbial genera were identified in the study. Bacteroidetes and Firmicutes were the two most dominant bacterial phyla in both cattle and buffaloes. However, there was no difference in the abundance of Bacteroidetes and Firmicutes in the rumen metagenome of cattle and buffaloes. Based on the abundance, the Proteobacteria was the 3rd largest phylum in the metagenome, constituting 18-20% in both host species. Euryarchaeota was the most abundant phylum of the methanogens, whereas Methanobacteriales and Methanobrevibacter were the most abundant orders and genera in both species. The methanogen abundances were not different between the two host species. Like the metagenome, the difference between the compositional and functional abundances (metagenome vs. metatranscriptome) of the Bacteroidetes and Firmicutes was not significant, whereas the proteobacteria were functionally less active than their metagenomic composition. Contrary to the metagenome, the Euryarchaeota was the 3rd most functional phylum in the rumen and constituted ~15% of the metatranscriptome. Methanobacteriales were the most functional methanogens, accounting for more than 2/3rd of the total archaeal functionality. These results indicated that the methanogens from Euryarchaeota were functionally more active as compared to their compositional abundance. The CH4 yield (g/kg DMI), CH4 emission (g/kg DDM), dry matter (DM) intake, and rumen fermentation did not vary between the two host species. Overall, the study established a substantial difference between the compositional abundances and metabolic functionality of the rumen microbiota; however, feeding cattle and buffaloes on the same diet resulted in similar microbiota composition, metabolic functionality, and CH4 yield. Further studies are warranted to investigate the effect of different diets and environments on the composition and metabolic functionality of the rumen microbiota.

Effects of biochar-amended soils as intermediate covers on the physical, mechanical and biochemical behaviour of municipal solid wastes.

The effects of biochar-amended soils as landfill covers have been extensively studied in terms of liquid and gas permeability. However, the influences of biochar-amended soils on the performance of municipal solid wastes (MSWs) in bioreactor landfills have not been well understood. This paper investigates the potential application of biochar-amended soils as final and intermediate covers in landfills. The MSWs with biochar-amended soils as final and intermediate covers were recirculated with mature leachate in laboratory-scale bioreactors. The pH, chemical oxygen demand, ammonia and volatile fatty acids (VFAs) concentrations of leachates, mass reduction rates, settlement, methane, and total gas generations of MSWs were investigated. The results indicate that biochar-amended soils as intermediate landfill covers can provide pH-buffer capacity, increase the pH of leachate and decrease the accumulation of VFAs in the early stage of decomposition. The concentration of ammonia in the leachate with biochar-amended soils as intermediate cover is lower than that with natural soils. The application of biochar-amended soils as intermediate and/or final covers increases the biocompression ratios and settlement of MSWs. The application of biochar-amended soils as final cover slightly decreases the methane generation potential (L0). Biochar-amended soils as intermediate covers increase L0 by 10%, and biochar-amended soils as both intermediate and final covers enhance L0 by 25%. The increase in the ammonia removal, settlement, and methane yield indicates the viability of biochar-amended soils as intermediate landfill covers. Further studies can focus on the long-term behaviour of MSWs with soil covers with different biochar amendment rates and particle sizes.

Monitoring of a microbial community during bioaugmentation with hydrogenotrophic methanogens to improve methane yield of an anaerobic digestion process.

Methane production by microbial fermentation of municipal waste is a challenge for better yield processes. This work describes the characterization of a hydrogenotrophic methanogen microbial community used in a bioaugmentation procedure to improve the methane yield in a thermophilic anaerobic process, digesting the organic fraction of municipal solid waste. The performance of the bioaugmentation was assessed in terms of methane production and changes in the microbial community structure. The results showed that bioaugmentation slightly improved the cumulative methane yield (+ 4%) in comparison to the control, and its use led to an acceleration of the methanogenesis stage. We observed associated significant changes in the relative abundance of taxa and their interactions, using high throughput DNA sequencing of V3-16S rRNA gene libraries, where the abundance of the archaeal hydrogenotrophic genus Methanoculleus (class Methanomicrobia, phylum Euryarchaeota) and the bacterial order MBA08 (class Clostridia, phylum Firmicutes) were dominant. The relevant predicted metabolic pathways agreed with substrate degradation and the anaerobic methanogenic process. The purpose of the study was to evaluate the effect of the addition of hydrogenotrophic methanogens in the generation of methane, while treating organic waste through anaerobic digestion.

Achieving efficient methane production from protein-rich organic waste in anaerobic digestion: Using conductive materials or regulating inoculum-to-substrate ratios?

The contribution of inoculum-to-substrate ratios (ISRs) and conductive materials (CMs) on the productivity of anaerobic digestion (AD) remains unclear, particularly for protein-rich organic waste. This study investigated whether the addition of CMs, i.e., biochar and iron powder, can overcome the limitations imposed by varying ISRs for the AD of protein as the sole substrate. Results indicate the ISR plays a decisive role in hydrolysis, acidification, and methanogenesis for protein conversion, irrespective of CMs addition. Methane production increased stepwise as the ISR escalated to 3:1. The addition of CMs provided limited improvement, and iron powder even inhibited methanogenesis at a low ISR. Bacterial community variations were contingent on the ISR, while iron powder supplementation significantly elevates the proportion of hydrogenotrophic methanogen. This study demonstrates that the addition of CMs could affect methanogenic efficiency but can not overcome the limitation of ISRs for the AD of protein.