The mechanisms of pH regulation on promoting volatile fatty acids production from kitchen waste.

The anaerobic acid production experiments were conducted with the pretreated kitchen waste under pH adjustment. The results showed that pH 8 was considered to be the most suitable condition for acid production, especially for the formation of acetic acid and propionic acid. The average value of total volatile fatty acid at pH 8 was 8814 mg COD/L, 1.5 times of that under blank condition. The average yield of acetic acid and propionic acid was 3302 mg COD/L and 2891 mg COD/L, respectively. The activities of key functional enzymes such as phosphotransacetylase, acetokinase, oxaloacetate transcarboxylase and succinyl-coA transferase were all enhanced. To further explore the regulatory mechanisms within the system, the distribution of microorganisms at different levels in the fermentation system was obtained by microbial sequencing, results indicating that the relative abundances of Clostridiales, Bacteroidales, Chloroflexi, Clostridium, Bacteroidetes and Propionibacteriales, which were great contributors for the hydrolysis and acidification, increased rapidly at pH 8 compared with the blank group. Besides, the proportion of genes encoding key enzymes was generally increased, which further verified the mechanism of hydrolytic acidification and acetic acid production of organic matter under pH regulation.

Potential and challenge in accelerating high-value conversion of CO2 in microbial electrosynthesis system via data-driven approach.

Microbial electrosynthesis for CO2 utilization (MESCU) producing valuable chemicals with high energy density has garnered attention due to its long-term stability and high coulombic efficiency. The data-driven approaches offer a promising avenue by leveraging existing data to uncover the underlying patterns. This comprehensive review firstly uncovered the potentials of utilizing data-driven approaches to enhance high-value conversion of CO2 via MESCU. Firstly, critical challenges of MESCU advancing have been identified, including reactor configuration, cathode design, and microbial analysis. Subsequently, the potential of data-driven approaches to tackle the corresponding challenges, encompassing the identification of pivotal parameters governing reactor setup and cathode design, alongside the decipheration of omics data derived from microbial communities, have been discussed. Correspondingly, the future direction of data-driven approaches in assisting the application of MESCU has been addressed. This review offers guidance and theoretical support for future data-driven applications to accelerate MESCU research and potential industrialization.

Biodegradation of organosulfur with extra carbon source: Insights into biofilm formation and bacterial metabolic processes.

Organosulfur compounds are prevalent in wastewater, presenting challenges for biodegradation, particularly in low-carbon environments. Supplementing additional carbon sources not only provides essential nutrients for microbial growth but also serves as regulators, influencing adaptive changes in biofilm and enhancing the survival of microorganisms in organosulfur-induced stress bioreactors. This study aims to elucidate the biodegradation of organosulfur under varying carbon source levels, placing specific emphasis on functional bacteria and metabolic processes. It has been observed that higher levels of carbon supplementation led to significantly improved total sulfur (TS) removal efficiencies, exceeding 83 %, and achieve a high organosulfur CH3SH removal efficiency of ~100 %. However, in the reactor with no external carbon source added, the oxidation end-product SO42- accumulated significantly, surpassing 120 mEq/m2-day. Furthermore, the TB-EPS concentration consistently increasedwith the ascending glucose concentration. The analysis of bacterial community reveals the enrichment of functional bacteria involved in sulfur metabolism and biofilm formation (e.g. Ferruginibacter, Rhodopeudomonas, Gordonia, and Thiobacillus). Correspondingly, the gene expressions related to the pathway of organosulfur to SO42- were notably enhanced (e.g. MTO increased by 27.7 %). In contrast, extra carbon source facilitated the transfer of organosulfur into amino acids in sulfur metabolism and promoted assimilation. These metabolic insights, coupled with kinetic transformation results, further validate distinct sulfur pathways under different carbon source conditions. The intricate interplay between bacteria growth regulation, pollutant biodegradation, and microbial metabolites underscores a complex network relationship that significantly contributes to efficient operation of bioreactors.

Chlorinated organophosphorus flame retardants induce the propagation of antibiotic resistance genes in sludge fermentation systems: Insight of chromosomal mutation and microbial traits.

Waste activated sludge (WAS) is a critical reservoir for antibiotic resistance genes (ARGs) due to the prevalent misuse of antibiotics. Horizontal gene transfer (HGT) is the primary mechanism for ARGs spread through mobile genetic elements (MGEs). However, the role of non-antibiotic organophosphorus flame retardants (Cl-OFRs) in ARG transmission in the WAS fermentation system remains unclear. This study examines the effects of tris(2-chloroethyl) phosphate (TCEP), a representative Cl-OFR, on ARG dynamics in WAS fermentation using molecular docking and metagenomic analysis. The results showed a 33.4 % increase in ARG abundance in the presence of TCEP. Interestingly, HGT did not appear to be the primary mechanism of ARG dissemination under TCEP stress, as evidenced by a 2.51 % decrease in MGE abundance. TCEP binds to sludge through hydrogen bonds with a binding energy of - 3.6 kJ/mol, leading to microbial damage and an increase in the proportion of non-viable cells. This interaction prompts a microbial shift toward Firmicutes with thick cell walls, which are significant ARG carriers. Additionally, TCEP induces chromosomal mutations through oxidative stress and the SOS response, contributing to ARG formation. Microorganisms also develop multidrug resistance mechanisms to expel TCEP and mitigate its toxicity. This study provides a comprehensive understanding of Cl-OFRs effects on the ARGs fates in WAS fermentation system and offers guidance for the safe and efficient treatment of Cl-OFRs and WAS.

N2O recovery from wastewater and flue gas via microbial denitrification: Processes and mechanisms.

Nitrous oxide (N2O) is increasingly regarded as a significant greenhouse gas implicated in global warming and the depletion of the ozone layer, yet it is also recognized as a valuable resource. This paper comprehensively reviews innovative microbial denitrification techniques for recovering N2O from nitrogenous wastewater and flue gas. Critical analysis is carried out on cutting-edge processes such as the coupled aerobic-anoxic nitrous decomposition operation (CANDO) process, semi-artificial photosynthesis, and the selective utilization of microbial strains, as well as flue gas absorption coupled with heterotrophic/autotrophic denitrification. These processes are highlighted for their potential to facilitate denitrification and enhance the recovery rate of N2O. The review integrates feasible methods for process control and optimization, and presents the underlying mechanisms for N2O recovery through denitrification, primarily achieved by suppressing nitrous oxide reductase (Nos) activity and intensifying competition for electron donors. The paper concludes by recognizing the shortcomings in existing technologies and proposing future research directions, with an emphasis on prioritizing the collection and utilization of N2O while considering environmental sustainability and economic feasibility. Through this review, we aim to inspire interest in the recovery and utilization of N2O, as well as the development and application of related technologies.

Combined transcriptomics and metabolomics to analyse the response of Cuminum cyminum L. under Pb stress.

Lead (Pb) can disrupt plant gene expression, modify metabolite contents, and influence the growth of plants. Cuminum cyminum L. is highly adaptable to adversity, but molecular mechanism by which it responds to Pb stress is unknown. For this study, transcriptomic and metabolomic sequencing was performed on root tissues of C. cyminum under Pb stress. Our results showed that high Pb stress increased the activity of peroxidase (POD), the contents of malondialdehyde (MDA) and proline by 80.03 %, 174.46 % and 71.24 %, respectively. Meanwhile, Pb stress decreased the activities of superoxide dismutase (SOD) and catalase (CAT) as well as contents of soluble sugars and GSH, which thus affected the growth of C. cyminum. In addition, Pb stress influenced the accumulation and transport of Pb in C. cyminum. Metabolomic results showed that Pb stress affected eight metabolic pathways involving 108 differentially expressed metabolites, primarily amino acids, organic acids, and carbohydrates. The differentially expressed genes identified through transcriptome analysis were mainly involved the oxidation reductase activity, transmembrane transport, phytohormone signaling, and MAPK signaling pathway. The results of this study will help to understand the molecular mechanisms of C. cyminum response to Pb stress, and provide a basis for screening seeds with strong resistance to heavy metals.

Efficient nitrate and Cr(VI) removal by denitrifier: The mechanism of S. oneidensis MR-1 promoting electron production, transportation and consumption.

When Cr(VI) and nitrate coexist, the efficiency of both bio-denitrification and Cr(VI) bio-reduction is poor because chromate hinders bacterial normal functions (i.e., electron production, transportation and consumption). Moreover, under anaerobic condition, the method about efficient nitrate and Cr(VI) removal remained unclear. In this paper, the addition of Shewanella oneidensis MR-1 to promote the electron production, transportation and consumption of denitrifier and cause an increase in the removal of nitrate and Cr(VI). The efficiency of nitrate and Cr(VI) removal accomplished by P. denitrificans as a used model denitrifier increased respectively from 51.3% to 96.1% and 34.3% to 99.8% after S. oneidensis MR-1 addition. The mechanism investigations revealed that P. denitrificans provided S. oneidensis MR-1 with lactate, which was utilized to secreted riboflavin and phenazine by S. oneidensis MR-1. The riboflavin served as coenzymes of cellular reductants (i.e., thioredoxin and glutathione) in P. denitrificans, which created favorable intracellular microenvironment conditions for electron generation. Meanwhile, phenazine promoted biofilm formation, which increased the adsorption of Cr(VI) on the cell surface and accelerated the Cr(VI) reduction by membrane bound chromate reductases thereby reducing damage to other enzymes respectively. Overall, this strategy reduced the negative effect of chromate, thus improved the generation, transportation, and consumption of electrons. SYNOPSIS: The presence of S. oneidensis MR-1 facilitated nitrate and Cr(VI) removal by P. denitrificans through decreasing the negative effect of chromate due to the metabolites' secretion.

Constructing Amorphous-Crystalline Interfacial Bifunctional Site Island-Sea Synergy by Morphology Engineering Boosts Alkaline Seawater Hydrogen Evolution.

The development of efficient and durable non-precious hydrogen evolution reaction (HER) catalysts for scaling up alkaline water/seawater electrolysis is highly desirable but challenging. Amorphous-crystalline (A-C) heterostructures have garnered attention due to their unusual atomic arrangements at hetero-interfaces, highly exposed active sites, and excellent stability. Here, a heterogeneous synthesis strategy for constructing A-C non-homogeneous interfacial centers of electrocatalysts on nanocages is presented. Isolated PdCo clusters on nanoscale islands in conjunction with Co3S4 A-C, functioning as a bifunctional site "island-sea" synergy, enable the dynamic confinement design of metal active atoms, resulting in excellent HER catalytic activity and durability. The hierarchical structure of hollow porous nanocages and nanoclusters, along with their large surface area and multi-dimensional A-C boundaries and defects, provides the catalyst with abundant active centers. Theoretical calculations demonstrate that the combination of PdCo and Co3S4 regulates the redistribution of interface electrons effectively, promoting the sluggish water-dissociation kinetics at the cluster Co sites. Additionally, PdCo-Co3S4 heterostructure nanocages exhibit outstanding HER activity in alkaline seawater and long-term stability for 100 h, which can be powered by commercial silicon solar cells. This finding significantly advances the development of alkaline seawater electrolysis for large-scale hydrogen production.

Emerging Two-Dimensional Carbonaceous Materials for Electrocatalytic Energy Conversions: Rational Design of Active Structures through High-Temperature Chemistry.

Electrochemical energy conversion and storage technologies involving controlled catalysis provide a sustainable way to handle the intermittency of renewable energy sources, as well as to produce green chemicals/fuels in an ecofriendly manner. Core to such technology is the development of efficient electrocatalysts with high activity, selectivity, long-term stability, and low costs. Here, two-dimensional (2D) carbonaceous materials have emerged as promising contenders for advancing the chemistry in electrocatalysis. We review the emerging 2D carbonaceous materials for electrocatalysis, focusing primarily on the fine engineering of active structures through thermal condensation, where the design, fabrication, and mechanism investigations over different types of active moieties are summarized. Interestingly, all the recipes creating two-dimensionality on the carbon products also give specific electrocatalytic functionality, where the special mechanisms favoring 2D growth and their consequences on materials functionality are analyzed. Particularly, the structure-activity relationship between specific heteroatoms/defects and catalytic performance within 2D metal-free electrocatalysts is highlighted. Further, major challenges and opportunities for the practical implementation of 2D carbonaceous materials in electrocatalysis are summarized with the purpose to give future material design guidelines for attaining desirable catalytic structures.

Transcriptomic Insights into Metabolism-Dependent Biosynthesis of Bacterial Nanocellulose.

Bacterial nanocellulose (BNC) is an attractive green-synthesized biomaterial for biomedical applications and various other applications. However, effective engineering of BNC production has been limited by our poor knowledge of the related metabolic processes. In contrast to the traditional perception that genome critically determines biosynthesis behaviors, here we discover that the glucose metabolism could also drastically affect the BNC synthesis in Gluconacetobacter hansenii. The transcriptomic profiles of two model BNC-producing strains, G. hansenii ATCC 53582 and ATCC 23769, which have highly similar genomes but drastically different BNC yields, were compared. The results show that their BNC synthesis capacities were highly related to metabolic activities such as ATP synthesis, ion transport protein assembly, and carbohydrate metabolic processes, confirming an important role of metabolism-related transcriptomes in governing the BNC yield. Our findings provide insights into the microbial biosynthesis behaviors from a transcriptome perspective, potentially guiding cellular engineering for biomaterial synthesis.

Simultaneous volatile fatty acids promotion and antibiotic resistance genes reduction in fluoranthene-induced sludge alkaline fermentation: Regulation of microbial consortia and cell functions.

The impact and mechanism of fluoranthene (Flr), a typical polycyclic aromatic hydrocarbon highly detected in sludge, on alkaline fermentation for volatile fatty acids (VFAs) recovery and antibiotic resistance genes (ARGs) transfer were studied. The results demonstrated that VFAs production increased from 2189 to 4272 mg COD/L with a simultaneous reduction of ARGs with Flr. The hydrolytic enzymes and genes related to glucose and amino acid metabolism were provoked. Also, Flr benefited for the enrichment of hydrolytic-acidifying consortia (i.e., Parabacteroides and Alkalibaculum) while reduced VFAs consumers (i.e., Rubrivivax) and ARGs potential hosts (i.e., Rubrivivax and Pseudomonas). Metagenomic analysis indicated that the genes related to cell wall synthesis, biofilm formation and substrate transporters to maintain high VFAs-producer activities were upregulated. Moreover, cell functions of efflux pump and Type IV secretion system were suppressed to inhibit ARGs proliferation. This study provided intrinsic mechanisms of Flr-induced VFAs promotion and ARGs reduction during alkaline fermentation.

Comprehensive analysis of bisphenol analogues in complex water using a group-targeting aptamer engineered by base mutation.

Bisphenol analogues (BPs) are typical environmental hormones with endocrine-disrupting effects and reproductive toxicity requiring analysis and monitoring in complex aquatic environments. However, the presence of various co-existing contaminants makes the accurate determination of total BPs difficult. To address this challenge, there is a strong need to obtain a group-targeting binder to specifically detect a class of BPs. In this work, for the first time we have identified the group-targeting BPs-aptamer with similar affinities for multiple structurally and qualitatively similar BPs. Base mutations were introduced into an aptamer specific to bisphenol A (BPA) and utilized molecular docking calculations to identify a group-targeting aptamer capable of binding BPs, including BPA, bisphenol B (BPB), bisphenol E (BPE) and bisphenol F (BPF) with binding constants in the range of 2.0 × 106 ∼ 2.7 × 106 / M. In addition, an electrochemical aptamer-based sensor (aptasensor) was constructed for highly sensitive and comprehensive analysis of a class of BPs. This aptasensor demonstrated remarkable anti-interference performance against co-existing contaminants at concentrations up to 100-fold and achieved an impressive detection limit of 6.7 pM. This innovative approach of engineering a group-targeting BPs-aptamer is important for the comprehensive analysis of BPs, providing insights into identification and monitoring a class of pollutants.

Mechanisms, methods and applications of machine learning in bio-alcohol production and utilization: A review.

Bio-alcohols have been proven promising alternatives to fossil fuels. Machine learning (ML), as an analytical tool for uncovering intrinsic correlations and mining data connotations, is also becoming widely used in the field of bio-alcohols. This article reviews the mechanisms, methods, and applications of ML in the bio-alcohols field. In terms of mechanisms, we describe the workflow of ML applications, emphasizing the importance of a well-defined research problem and complete feature engineering for a robust model. Prediction and optimization are the main application scenarios. In terms of methods, we illustrate the characteristics of different ML models and analyze their applicability in the bio-alcohol field. The role of ML in the production of bio-methanol by pyrolysis and gasification, as well as in the three stages of fermentation for bioethanol production are highlighted. In terms of utilization, ML is used to optimize engine performance and reduce emissions. This review provides guidance on how to use novel ML methods in the bio-alcohol field, showing the potential of ML to streamline work in the whole biofuel field.

Valorization of protein-rich waste and its application.

Energy shortages present significant challenges with the rising population and dramatic urbanization development. The effective utilization of high-value products generated from massive protein-rich waste has emerged as an excellent solution for mitigating the growing energy crisis. However, the traditional disposal and treatment of protein-rich waste, have been proven to be ineffective in resource utilization, which led to high chemical oxygen demand and water eutrophication. To effectively address this issue, hydrolysate and bioconversion products from protein-rich waste have been widely investigated. Herein, we aim to provide an overview of the valorization of protein-rich waste based on a comprehensive analysis of publicly available literature. Firstly, the sources of protein-rich waste with various quantities and qualities are systematically summarized. Then, we scrutinize and analyze the hydrolysis approaches of protein-rich waste and the versatile applications of hydrolyzed products. Moreover, the main factors influencing protein biotransformation and the applications of bioconversion products are covered and extensively discussed. Finally, the potential prospects and future directions for the valorization of protein-rich waste are proposed pertinently.

Nitrogen-doped graphene for tetracycline removal via enhancing adsorption and non-radical persulfate activation.

Nitrogen-doped graphene (NG) was synthesized via direct thermal annealing treatment. The obtained NG showed outstanding removal ability for tetracycline (TC) ascribed to enhanced adsorption and persulfate activation. The maximum TC adsorption capacity calculated from the Langmuir model of NG was 227.3 mg/g, which was 1.66 times larger than nitrogen-free graphene. The coexistence of NG and persulfate (PS) exhibited complete degradation of TC within 120 min attributed to the successful modification of nitrogen. Further analysis demonstrated that non-radical electron transfer was the dominant degradation pathway, which was different from the widely acknowledgeable radical mechanism. An electron donor-mediator-acceptor system was introduced, in which TC, NG, and PS performed as electron donor, mediator, and acceptor, respectively. The potential intermediates in the TC degradation process were detected and toxicity assessment was also performed. In addition, more than 75.8% of total organic carbon was removed, and excellent reusability was manifested in multiple adsorption and degradation experiments.

Phenanthrene regulates metabolic pathways for hydrogen accumulation in sludge alkaline dark fermentation.

The influence of phenanthrene (PHE), a general polycyclic aromatic hydrocarbon in waste activated sludge, on sludge alkaline dark fermentation for hydrogen accumulation was investigated prospectively. The yield of hydrogen was 16.2 mL/g TSS with 50 mg/kg TSS PHE, which was 1.3-fold greater than that of the control. Mechanism research demonstrated that hydrogen production and the abundance of functional microorganisms were facilitated, whereas those of homoacetogenesis were reduced. The activity of pyruvate ferredoxin oxidoreductase in the conversion of pyruvate to reduced ferredoxin for hydrogen production was promoted by 57.2%, and that of carbon monoxide dehydrogenase and formyltetrahydrofolate synthetase, closely associated with hydrogen consumption, was suppressed by 60.5% and 55.9%, respectively. Moreover, the encoding genes involved in pyruvate metabolism were significantly up-regulated, while genes related to consuming hydrogen to reduce carbon dioxide and produce 5-methyltetrahydrofolate were down-regulated. This study notably illustrates the effect of PHE on hydrogen accumulation from metabolic pathways.

Electrosynthesis of Hydrogen Peroxide through Selective Oxygen Reduction: A Carbon Innovation from Active Site Engineering to Device Design.

Electrochemical synthesis of hydrogen peroxide (H2 O2 ) through the selective oxygen reduction reaction (ORR) offers a promising alternative to the energy-intensive anthraquinone method, while its success relies largely on the development of efficient electrocatalyst. Currently, carbon-based materials (CMs) are the most widely studied electrocatalysts for electrosynthesis of H2 O2 via ORR due to their low cost, earth abundance, and tunable catalytic properties. To achieve a high 2e- ORR selectivity, great progress is made in promoting the performance of carbon-based electrocatalysts and unveiling their underlying catalytic mechanisms. Here, a comprehensive review in the field is presented by summarizing the recent advances in CMs for H2 O2 production, focusing on the design, fabrication, and mechanism investigations over the catalytic active moieties, where an enhancement effect of defect engineering or heteroatom doping on H2 O2 selectivity is discussed thoroughly. Particularly, the influence of functional groups on CMs for a 2e- -pathway is highlighted. Further, for commercial perspectives, the significance of reactor design for decentralized H2 O2 production is emphasized, bridging the gap between intrinsic catalytic properties and apparent productivity in electrochemical devices. Finally, major challenges and opportunities for the practical electrosynthesis of H2 O2 and future research directions are proposed.

Valorization of food waste into short-chain fatty acids via enzymatic pretreatment: Effects of fermentation-pH on acid-producing processes and microbial metabolic functions.

Food waste (FW) has been widely considered as an essential resource for the production of short-chain fatty acids (SCFAs), an important class of chemicals with wide applications and over 20 million tons of annual market demand, by anaerobic fermentation. Although enzymatic pre-treatment could improve the FW biodegradation efficiency, resulting in enhanced efficiency of solubilization and hydrolysis, the influence of fermentation-pH on the SCFAs production and the metabolic functions, have rarely been reported. This study demonstrated that the uncontrolled pH could efficiently lead to an increase in the SCFAs production (33011 mgCOD/L) during long-term fermentation of FW (mainly consisting of 48.8% carbohydrates, 20.6% proteins, and 17.4% lipids) after enzymatic pre-treatment compared to the control (16413 mgCOD/L). Meanwhile, the acid-producing processes (i.e., solubilization, hydrolysis, and acidification) were synchronously enhanced by the enzymatic pre-treatment and no control over fermentation-pH. Metagenomic analysis revealed that the acid-forming microorganisms (i.e., Olsenella sp. and Sporanaerobacter) were significantly accumulated, and the corresponding genetic expressions related to extracellular hydrolysis (i.e., aspB and gltB), membrane transport (i.e., metL and glnH), and intracellular material metabolism (i.e., pfkA and ackA) were evidently stimulated, thereby promoting ultimate SCFAs generation. Although the alkaline conditions could further slightly increase the SCFAs yield slightly (37100 mgCOD/L) and also stimulate the metabolic activities, it might not be suitable for large-scale practical applications due to additional costs associated with alkaline chemical additives.

Metabolite Cross-Feeding Promoting NADH Production and Electron Transfer during Efficient SMX Biodegradation by a Denitrifier and S. oneidensis MR-1 in the Presence of Nitrate.

Antibiotics often coexist with other pollutants (e.g., nitrate) in an aquatic environment, and their simultaneous biological removal has attracted widespread interest. We have found that sulfamethoxazole (SMX) and nitrate can be efficiently removed by the coculture of a model denitrifier (Paracoccus denitrificans, Pd) and Shewanella oneidensis MR-1 (So), and SMX degradation is affected by NADH production and electron transfer. In this paper, the mechanism of a coculture promoting NADH production and electron transfer was investigated by proteomic analysis and intermediate experiments. The results showed that glutamine and lactate produced by Pd were captured by So to synthesize thiamine and heme, and the released thiamine was taken up by Pd as a cofactor of pyruvate and ketoglutarate dehydrogenase, which were related to NADH generation. Additionally, Pd acquired heme, which facilitated electron transfer as heme, was the important composition of complex III and cytochrome c and the iron source of iron sulfur clusters, the key component of complex I in the electron transfer chain. Further investigation revealed that lactate and glutamine generated by Pd prompted So chemotactic moving toward Pd, which helped the two bacteria effectively obtain their required substances. Obviously, metabolite cross-feeding promoted NADH production and electron transfer, resulting in efficient SMX biodegradation by Pd and So in the presence of nitrate. Its feasibility was finally verified by the coculture of an activated sludge denitrifier and So.

Proteomic profiling of robust acetoclastic methanogen in chrysene-altered anaerobic digestion: Global dissection of enzymes.

Methanogen is a pivotal player in pollution treatment and energy recovery, and emerging pollutants (EPs) frequently occur in methanogen-applied biotechnology such as anaerobic digestion (AD). However, the direct effect and underlying mechanism of EPs on crucial methanogen involved in its application still remain unclear. The positive effect of chrysene (CH) on semi-continuous AD of sludge and the robust methanogen was dissected in this study. The methane yield in the digester with CH (100 mg/kg dry sludge) was 62.1 mL/g VS substrate, much higher than that in the control (46.1 mL/g VS substrate). Both methane production from acetoclastic methanogenesis (AM) and the AM proportion in the methanogenic pathway were improved in CH-shaped AD. Acetoclastic consortia, especially Methanosarcina and functional profiles of AM were enriched by CH in favor of the corresponding methanogenesis. Further, based on pure cultivation exposed to CH, the methanogenic performance, biomass, survivability and activity of typical Methanosarcina (M. barkeri) were boosted. Notably, iTRAQ proteomics revealed that the manufacturing (transcription and translation), expression and biocatalytic activity of acetoclastic metalloenzymes, particularly tetrahydromethanopterin S-methyltransferase and methyl-coenzyme M reductase with cobalt/nickel-cofactor (F430 and cobalamin), and acetyl-CoA decarbonylase/synthase with cobalt/nickel-active site, of M. barkeri were upregulated significantly with fold changes in the range of 1.21-3.20 due to the CH presence. This study shed light on EPs-affecting industrially crucial methanogen at the molecular biology level during AD and had implications in the technical relevance of methanogens.