Anaerobic Digestion Production Research Articles

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.

Characterization and utilization of industrial wastewater in biorefinery systems: A comprehensive approach

AbstractThis review explores the innovative utilization of industrial effluents in biorefinery applications, addressing the environmental challenges posed by the complex mixture of pollutants in industrial wastewater. It emphasizes the transformation of these effluents into valuable resources, such as biofuels, biochemicals, and bioplastics, through advanced biotechnological processes including anaerobic digestion, fermentation, enzymatic conversion, and microbial biomass production. The study highlights the critical role of microbial biocatalysts in breaking down diverse pollutants and transforming waste into wealth, thereby contributing to sustainable industrial practices and a circular economy. Challenges such as variability in effluent composition, inhibitory substances, and the necessity for robust bioprocesses are discussed, along with suggestions for future research directions like effluent characterization, development of specialized microbial consortia, and effective monitoring and control strategies. The review underscores the importance of collaboration between industry, academia, and government to advance biorefinery technologies, ultimately advocating for a sustainable and resource‐efficient future through the innovative treatment of industrial wastewaters.

The Selenoproteome as a Dynamic Response Mechanism to Oxidative Stress in Hydrogenotrophic Methanogenic Communities.

Methanogenesis is a critical process in the carbon cycle that is applied industrially in anaerobic digestion and biogas production. While naturally occurring in diverse environments, methanogenesis requires anaerobic and reduced conditions, although varying degrees of oxygen tolerance have been described. Microaeration is suggested as the next step to increase methane production and improve hydrolysis in digestion processes; therefore, a deeper understanding of the methanogenic response to oxygen stress is needed. To explore the drivers of oxygen tolerance in methanogenesis, two parallel enrichments were performed under the addition of H2/CO2 in an environment without reducing agents and in a redox-buffered environment by adding redox mediator 9,10-anthraquinone-2,7-disulfonate disodium. The cellular response to oxidative conditions is mapped using proteomic analysis. The resulting community showed remarkable tolerance to high-redox environments and was unperturbed in its methane production. Next to the expression of pathways to mitigate reactive oxygen species, the higher redox potential environment showed an increased presence of selenocysteine and selenium-associated pathways. By including sulfur-to-selenium mass shifts in a proteomic database search, we provide the first evidence of the dynamic and large-scale incorporation of selenocysteine as a response to oxidative stress in hydrogenotrophic methanogenesis and the presence of a dynamic selenoproteome.

Parametric Evaluation of Biogas Production from Sawdust and Treated Human Faeces: Optimal Custom Design Approach

As energy demand is increasing astronomically, fossil-based fuels became scarce and more expensive, and carbon dioxide emission levels became of greater concern. Thus, the need to convert the co-digestion of sawdust and treated human faeces to produce biogas, a carbon-neutral gas to replace fossil fuels. From the outcome, the logistic function model performance was satisfactory in the simulation of the experimental process. The codigestion of sawdust and human faeces at 2.5 kg each produced the highest biogas output of 5.43 L using Optimal (Custom) Design of Mixture approach but the human faeces alone gave a better biogas quality. Additionally, the high coefficient of determination (R2) ranging between 0.9959 - 0.9879 and standard deviation of 0.023 - 0.321 for temperature - biogas yield respectively, fall within an ideal range. Thus, the model proved to be a useful tool in predicting anaerobic digestion and biogas production processes. Furthermore, the FTIR analysis of sawdust and human faeces indicates their potential to produce biogas if subjected to anaerobic environment. In addition, the GCMS analyzed the % of methane to be 58.40% which is within accepted range. The biogas was tested with a locally fabricated gas burner for flammability and it ignited with a blue flame. Thus, this experiment can be translated to an industrial scale biogas production for cooking and electricity to boost local economy and alleviate poverty.

Relation of organic fractions in fresh and stored fecal sludge and foodwaste to biogas production

Abstract There is limited understanding of the potential for anaerobic digestion and biogas production from fecal sludge. In this study, biomethane potential (BMP) tests from fresh, stored, and dewatered fecal sludge, together with co-digestion with fresh foodwaste, revealed that fresh fecal sludge produced similar cumulative biogas (CBG) to fresh foodwaste (615–627 mL/gVS), while stored fecal sludge showed a wide range of gas production (13–449 mL/gVS). Co-digestion significantly enhanced the CBG production of fresh (1.2×), dewatered (1.5×), and stored (29–36×) fecal sludge. In BMP tests with the higher range of gas production, a biphasic CBG production was observed, with degradation of readily biodegradable organics occurring during the first week. The first-order rate coefficients indicated hydrolysis limitation, which was also confirmed by the presence of slow-growing methanogens (Halobacterota). Priming with co-digestion significantly enhanced CBG from stored fecal sludge. The physical–chemical metrics VS/TS and TOC/TN were not predictors of biogas production, while BOD/COD and sCOD were better indicators, suggesting that metrics of stabilization representing biologically available fractions are more representative than metrics of entire pools of organic matter. This study suggests that biogas production from anaerobic digestion is viable for fresh fecal sludge, whereas for stored fecal sludge it requires co-treatment or pretreatment.

Enhancing Waste-to-Energy and Hydrogen Production through Urban–Industrial Symbiosis: A Multi-Objective Optimisation Model Incorporating a Bayesian Best-Worst Method

A surging demand for sustainable energy and the urgency to lower greenhouse gas emissions is driving industrial systems towards more eco-friendly and cost-effective models. Biogas from agricultural and municipal organic waste is gaining momentum as a renewable energy source. Concurrently, the European Hydrogen Strategy focuses on green hydrogen for decarbonising the industrial and transportation sectors. This paper presents a multi-objective network design model for urban–industrial symbiosis, incorporating anaerobic digestion, cogeneration, photovoltaic, and hydrogen production technologies. Additionally, a Bayesian best-worst method is used to evaluate the weights of the sustainability aspects by decision-makers, integrating these into the mathematical model. The model optimises industrial plant locations considering economic, environmental, and social parameters, including the net present value, energy consumption, and carbon footprint. The model’s functionalities are demonstrated through a real-world case study based in Emilia Romagna, Italy. It is subject to sensitivity analysis to evaluate how changes in the inputs affect the outcomes and highlights feasible trade-offs through the exploration of the ϵ-constraint. The findings demonstrate that the model substantially boosts energy and hydrogen production. It is not only economically viable but also reduces the carbon footprint associated with fossil fuels and landfilling. Additionally, it contributes to job creation. This research has significant implications, with potential future studies intended to focus on system resilience, plant location optimisation, and sustainability assessment.

A Case Study on Small- and Centralized Biogas Plants and Energy Production Capacities in South Gyeongsang Province

Objectives : The primary goals of this research were to assess the viability and practicality of small-scale village facilities as well as central commercial biogas plants. Additionally, the study aimed to create predictive models by exploring various codigestion scenarios.Methods : The study conducted a comprehensive analysis of available biomass and its maximum energy potential through anaerobic digestion in every city, county, and village within South Gyeongsang province. Five distinct codigestion scenarios were explored, encompassing assessments of processing capacity, energy production potential, and the necessary digester capacity for anaerobic digestion. At the village level, the scenarios comprised: C1, which involved sole digestion of manure; C2, codigestion of manure and food waste in a 7:3 ratio; C3, codigestion of pig slurry and slaughterhouse waste in a 9:1 ratio; C4, multiple codigestion with PS:FW:SW=6.5:2.8:0.8; and finally, C5, involving the addition of sewage and sewage sludge to the codigestion process of C4's biomass.Results and Discussion : The biomass generated in South Gyeongsang province was 9430 tons/day, with a methane production potential of 167 million cubic meters/year. This biomass had an energy production potential of 156,000 TOE/year and a potential electricity generation of 732.7 GW/year, based on the annual petroleum conversion ton. Codigestion (C5) enabled up to 720% more electricity generation compared to sole digestion of manure (C1). Mixing pig slurry and food waste in a 7:3 ratio resulted in approximately 18% more electricity production compared to the case where manure was mixed with slaughterhouse by-products in a 9:1 ratio.Conclusion : Biomass imbalance was significant in most regions, particularly due to high variations in food waste generation between regions. Obtaining alternative resources and integrating various biomass for anaerobic digestion, especially in rural areas, is crucial for achieving stable anaerobic digestion and high methane production. Regions with high biomass density are predicted to support large-scale biogas facilities following European standards, while 25 villages showed the potential for small-scale biogas facilities.

Composite nanofiber membranes to enhance the performance of high solids anaerobic digestion of organic rural household waste resources

This study investigated the effect of using nanofiber membrane composites containing Prussian blue-like compound nanoparticles (PNPs) with a stainless steel mesh as the carrier (NMCs) to relieve ammonia nitrogen inhibition of rural organic household waste during high-solid (total solid content of 15 %) anaerobic digestion (AD) and increase methane production. NMCs with different PNP contents were added to high-solid AD. When NMC with 15 % PNPs was added, the concentrations of volatile fatty acids and ammonia nitrogen were lower than those of the blank control group, and the methane yield was the highest (301.31 mL/g volatile solids [VS]), at 2.8 times higher than that of the blank control group. The R2 value of the modified Gompertz equation was 0.997. During digestion, the activities of extracellular protease and coenzyme F420 were higher than those of the control group. NMCs were successfully recovered at the end of the experiment, and confocal scanning three-dimensional electron micrographs indicated that NMC with 15 % PNPs of NMCs had stronger biological activity; microbial community structure analysis indicated increased abundance of hydrogenotrophic methanogens in this group. The study provides a new solution for improving the efficiency of biogas production and the recycling of exogenously added materials.

Effect of Antibiotics and Thermophilic Pre-Treatment on Anaerobic Co-Digestion of Pig Manure and Corn Straw

The excessive use of antibiotics in the pig breeding industry leads to the accumulation of antibiotic residuals in the environment, which is attributed to the improper treatment of livestock excrements. Sulfamethoxazole (SMX) and Norfloxacin (NOR) are antibiotics used in pig breeding and veterinary medicines as growth promoters and antibacterial drugs. The aim of this study was to examine the effects of these antibiotics and thermophilic pre-treatment on methane (CH4) yield by anaerobic co-digestion (AcoD) of pig manure and corn straw under mesophilic temperature condition (35 ± 1 °C). We used antibiotics at a concentration of 0, 20 and 60 mg L−1 (three stages) in two lab-scale continuously stirred tank reactors. The first reactor was run using untreated fed and the second reactor was run with thermophilic (55 ± 1 °C) pre-treated fed. The results showed that the CH4 productions from reactor one were 205, 163 and 128 and from reactor two were 222, 185 and 173 mL CH4 g−1 VSadded, respectively. This elucidates that the presence of antibiotics had a negative effect on CH4 production. Moreover, thermophilic pre-treatment improved the performance of the anaerobic digestion and CH4 production.

Pretreatment and Bioconversion for Valorization of Residues of Non-Edible Oilseeds

Biodiesel production currently follows a first-generation model using edible oils as raw materials. Such a production model is unsustainable, considering that it is limited by the high cost of edible oils, competes with the food sector, and is linked to deforestation and other environmental threats. Changing the raw material base to non-edible oils provides an opportunity to increase the sustainability of the biodiesel industry and to avoid conflicts with food production. Processing non-edible oilseeds for extracting the oil to be used for producing biodiesel generates large amounts of residues, such as de-oiled cakes, seed husks, and fruit shells and pods as well as plant stems and leaves resulting from pruning and other agronomy practices. Most of those residues are currently disposed of by burning or used in a suboptimal way. Bioconversion following the sugar platform route, anaerobic digestion, or enzyme production provides means for upgrading them to advanced biofuels and high-added value products. Bioconversion of plant biomass, including oilseed residues, requires pretreatment to enhance their susceptibility to enzymes and microorganisms. This review provides an outlook on bioconversion approaches applicable to different residues of oilseed-bearing plant species. Recent reports on the pretreatment of non-edible oilseed residues for enhancing their bioconversion through either the sugar platform route or anaerobic digestion are critically discussed. This review is based on an exhaustive Web of Science search performed in January–May 2023.

Benefits of pretreated water hyacinth for enhanced anaerobic digestion and biogas production

Benefits of pretreated water hyacinth for enhanced anaerobic digestion and biogas production

Preparation and Application of Carbon-Based Materials in the Production of Medium-Chain Carboxylic Acids by Anaerobic Digestion: A Review

The main purpose of this article is to explore the mechanism of action of carbon-based materials in the anaerobic digestion (AD) production of medium-chain carboxylic acids (MCCA). Currently, there are various methods to increase production, but there is no review on how carbon-based materials improve MCCA. This paper first introduced the chain elongation (CE) technology, focusing on the factors affecting the production of MCCA by AD, such as pH, temperature, the ratio of electron donor (ED) to an electron acceptor (EA), substrate type, and other related factors. This article introduces the preparation and characteristics of carbon-based materials, as well as the effect and mechanism of adding carbon-based materials to AD acid production. Finally, the shortcomings of the current research were pointed out, and future research directions were prospected, aiming to provide a reference for improving the efficiency of AD of MCCA using carbon-based materials.

Iron zero valent nanoparticles applied in the biorefinery concept: Functional genes in continuous bioreactor fermenting vinasse

Increased production of biogas and volatile acids was evaluated by the addition of Fe0 Nanoparticles (NP) on Anaerobic Fluidized Bed Reactor (AFBR-NP) with expanded clay as support material (45.1 mg Fe0 g−1 expanded clay), fermenting vinasse, and operated at 37 °C and 3h Hydraulic Retention Time (HRT). Higher H2 percentage in the biogas composition was observed in AFBR-NP (50–65%) when compared to AFBR without NP, AFBR-Control (24–43%). A 1.2-fold higher valeric acid production was also observed as a result of NPs supplementation. Genes related to the first steps of vinasse anaerobic degradation in AFBR-NP (beta-glucosidase, glucokinase and pyruvate-ferredoxin oxidoreductase) and the genes related to volatile acids production (L- and d-lactate dehydrogenase, 3-hydroxybutyryl-CoA dehydrogenase, acetate kinase, pyruvate kinase and fumarate reductase flavoprotein) were observed by metagenome sequencing. A 4.2-fold higher relative abundance of Secundilactobacillus genus in AFBR-NP was evidenced. The genetic and microbial community differences observed with the addition of NP resulted in improved biorefinery products of anaerobic digestion.

From Microalgae to Bioenergy: Recent Advances in Biochemical Conversion Processes

Concerns about rising energy demand, fossil fuel depletion, and global warming have increased interest in developing and utilizing alternate renewable energy sources. Among the available renewable resources, microalgae biomass, a third-generation feedstock, is promising for energy production due to its rich biochemical composition, metabolic elasticity, and ability to produce numerous bioenergy products, including biomethane, biohydrogen, and bioethanol. However, the true potential of microalgae biomass in the future bioenergy economy is yet to be realized. This review provides a comprehensive overview of various biochemical conversion processes (anaerobic digestion, direct biophotolysis, indirect biophotolysis, photo fermentation, dark fermentation, microalgae-catalyzed photo fermentation, microalgae-catalyzed dark fermentation, and traditional alcoholic fermentation by ethanologenic microorganisms) that could be adapted to transform microalgae biomass into different bioenergy products. Recent advances in biochemical conversion processes are compiled and critically analyzed, and their limitations in terms of process viability, efficacy, scalability, and economic and environmental sustainability are highlighted. Based on the current research stage and technological development, biomethane production from anaerobic digestion and bioethanol production from traditional fermentation are identified as promising methods for the future commercialization of microalgae-based bioenergy. However, significant challenges to these technologies’ commercialization remain, including the high microalgae production costs and low energy recovery efficiency. Future research should focus on reducing microalgae production costs, developing an integrated biorefinery approach, and effectively utilizing artificial intelligence tools for process optimization and scale-up to solve the current challenges and accelerate the development of microalgae-based bioenergy.

High-throughput profiling the effects of zinc on antibiotic resistance genes in the anaerobic digestion of swine manure

ABSTRACT The problem of antibiotic resistance genes (ARGs) caused by heavy metals has attracted extensive attention of human beings. Zn, a widely used feed additive, has a very high residue in swine manure, but the distribution characteristics of ARGs imposed by Zn in anaerobic digestion (AD) products are not clear. In this study, the behaviour of mobile genetic elements (MGEs), bacterial community, and their association with ARGs were determined in the presence of 125 and 1250 mg L–1 Zn in AD system of swine manure. Zn-treated enriched the abundance of ARGs, and produced some new genotypes that were not detected in CK treatment. In addition, low concentration of Zn significantly increased the relative abundance of ARGs, as compared to higher Zn and CK group. Correspondingly, the abundances of most top30 genus were highest in ZnL (125 mg L–1 Zn), followed by CK and ZnH (1250 mg L–1 Zn). Notably, network analysis showed that the relationship between ARGs and MGEs is closer than that ARGs and bacteria, suggesting that ARGs increased in Zn-treated, especially low level Zn, may be due to the amplification transfer of ARGs among varied microorganisms by horizontal transfer with MGEs. Therefore, strengthen the management of in livestock manure is crucial to control the spread of ARGs in organic fertilizers.

Optimizing food waste biodigestion: pre-selection of microbial groups and isolated enzymes using the plackett-burman experimental design

This study investigated the use of anaerobic biodigestion as an ecological technique for treating organic waste in urban solid waste. Biogas production is carried out through the action of microorganisms, and the use of enzymatic hydrolysis can increase biogas production, although it presents economic challenges. The aim of the study was to pre-select microbiological groups and enzymes to optimize the process and increase biogas production using statistical tools. Organic waste from a restaurant and sludge from a sewage treatment plant in Brazil were used, and three bacterial strains and 12 groups of microorganisms, as well as commercial enzymes, were evaluated. The study was divided into four stages to reduce the number of independent variables and increase the accuracy of methane and biogas production results. The results indicated that careful selection of microbial groups had a significant positive impact on biogas and methane production. The interaction between pre-selected groups of microorganisms and enzymes resulted in significant positive effects on cumulative biogas and biometane production. The study concluded that careful selection of microbial groups is essential to maximize biogas production and the efficiency of the anaerobic digestion process. Further, a more detailed identification of microorganisms and their interactions is needed to further optimize biogas production. In general, the appropriate choice of microbial groups is fundamental to the success of anaerobic digestion and biogas production.

Potential Greenhouse Gas Mitigation from Utilising Pig Manure and Grass for Hydrothermal Carbonisation and Anaerobic Digestion in the UK, EU, and China

Pig manure currently results in sizeable greenhouse gas emissions, during storage and spreading to land. Anaerobic digestion and hydrothermal carbonisation could provide significant greenhouse gas mitigation, as well as generate renewable heat and power (with anaerobic digestion), or a peat-like soil amendment product (with hydrothermal carbonisation). The greenhouse gas mitigation potential associated with avoidance of pig manure storage and spreading in the UK, EU, and China, as well as the potential to provide heat and power by anaerobic digestion and soil amendment products by hydrothermal carbonisation was herein determined. In each case, the mono-conversion of pig manure is compared to co-conversion with a 50:50 mixture of pig manure with grass. Anaerobic digestion displayed a greater greenhouse gas mitigation potential than hydrothermal carbonisation in all cases, and co-processing with grass greatly enhances greenhouse gas mitigation potential. China has the largest greenhouse gas mitigation potential (129 MT CO2 eq), and greatest mitigation per kg of pig manure (1.8 kgCO2/kg pig manure volatile solids). The energy grid carbon intensity has a significant impact on the greenhouse gas mitigation potential of the different approaches in the different regions. Pig manure is generated in large amounts in China, and the energy generated from biogas offsets a higher carbon intensity grid. Greenhouse gas savings from the anaerobic digestion of pig manure and grass have been calculated to provide a significant potential for reducing total greenhouse gas emissions representation in China (1.05%), the EU (0.92%), and the UK (0.19%). Overall, the utilisation of pig manure could bring about substantial greenhouse savings, especially through co-digestion of pig manure with grass in countries with large pig farming industries and carbon intense energy mixes.

Development of Digested Sludge-Assimilating and Biohydrogen-Yielding Microflorae

Digested sludge (DS) is a waste product of anaerobic digestion that is produced during the biodegradation of excess sludge. It cannot be used as a substrate for further biogas production owing to its recalcitrant nature. In the present study, we used a heat treatment technique to convert DABYS microflora (DABYS = digested sludge-assimilating and biogas-yielding soil microflora), which degraded DS and produced methane gas, to a microflora that could produce hydrogen gas from DS. Heat treatment at 80 and 100 °C inactivated the methanogens that consume hydrogen for methane production but did not affect the thermotolerant bacteria. We developed three microflorae (DABYS-A80, DABYS-A100, and DABYS-80B) to exclusively produce hydrogen gas. They included representatives from the anaerobic eubacterial families Clostridiaceae and Enterobacteriaceae. Pseudomonas sp. was also present in DABYS-A80 and DABYS-A100. It is thought that bacteria in the Enterobacteriaceae family or Pseudomonas genus survive heat treatment because they are embedded in microgranules. Enzymatic analysis suggested that the microflorae hydrolyzed DS using cellulase, chitinase, and protease. Under optimum culture conditions, DABYS-A80, -A100, and B-100 produced gas yields of 8.0, 7.1, and 2.6 mL, respectively, from 1.0 g of dried DS.

Biomethane generation and CO2 recovery through biogas production using brewers' spent Grains

In Brazil approximately 13 billion liters of beer were produced in 2016. The massive production of beer goes hand in hand with the generation of a huge amount of waste during the production process; a greater part of this waste is composed of about 2.6 million tons of Brewers' Spent Grains (BSG) the main waste. Due to their organic characteristics and in order to reduce the environmental impacts caused by their improper disposal or treatment, these residues can be effectively treated in anaerobic systems. One of the products of anaerobic digestion is biogas; this gas contains methane (CH4) and dioxide carbon (CO2), and further other gases in lower concentrations; these include oxygen, hydrogen sulfide and hydrogen. When biogas is purified, it transforms into biomethane, which is rich in methane with high calorific value. The processes involving the purification of biogas lead to the simultaneous recovery of other gases, such as CO2 - which is used in the brewing industry. Using experimental assays, this work evaluates the biochemical methanogenic potential of BSG. The results obtained showed that nearly 82 m3CH4 t−1DM (Dry Matter) of biomethane can be produced from the substrate and about 29 m3CO2 t−1DM can be recovered from the substrate. Thus, beer production in Brazil can be used as a source of energy production and CO2 recovery. Our findings show that the reutilization of BSG can yield 76% of CO2 required in the brewing process; reducing in US $116 million in the costs incurred in the purchase of CO2.

Effect of graphene and graphene oxide on antibiotic resistance genes during copper-contained swine manure anaerobic digestion.

Copper is an important selectors for antibiotic resistance genes (ARGs) transfer because of metal-antibiotic cross-resistance and/or coresistance. Due to carbon-based materials' good adsorption capacity for heavy metals, graphene and graphene oxide have great potential to reduce ARGs abundance in the environment with copper pollution. To figure out the mechanics, this study investigated the effects of graphene and graphene oxide on the succession of ARGs, mobile genetic elements (MGEs), heavy metal resistance genes (HMRGs), and bacterial communities during copper-contained swine manure anaerobic digestion. Results showed that graphene and graphene oxide could reduce ARGs abundance in varying degrees with the anaerobic reactors that contained a higher concentration of copper. Nevertheless, graphene decreased the abundance of ARGs more effectively than graphene oxide. Phylum-level bacteria such as Firmicutes, Bacteroidetes, Spirochaetes, and Verrucomicrobiaat were significantly positively correlated with most ARGs. Network and redundancy analyses demonstrated that alterations in the bacterial community are one of the main factors leading to the changes in ARGs. Firmicutes, Bacteroidetes, and Spirochaetes were enriched lower in graphene reactor than graphene oxide in anaerobic digestion products, which may be the main reason that graphene is superior to graphene oxide in reduced ARGs abundance. Additionally, ARGs were close to HMRGs than MGEs in the treatments with graphene, the opposite in graphene oxide reactors. Therefore, we speculate that the reduction of HMRGs in graphene may contribute to the result that graphene is superior to graphene oxide in reduced ARGs abundance in anaerobic digestion.