# Ammonia inhibition in anaerobic digestion and weakly electrical stimulation strategies: Exploring mitigation mechanisms and economic benefits - A review

Jiao Li, Yuchen Zhang, Ruixiang Zou, Wenhui Sun, Wenhui An, Eric

Lichtfouse, Suyun Xu, Hongbo Liu

► To cite this version:

Jiao Li, Yuchen Zhang, Ruixiang Zou, Wenhui Sun, Wenhui An, et al.. Ammonia inhibition in anaerobic digestion and weakly electrical stimulation strategies: Exploring mitigation mechanisms and economic benefits - A review. Bioresource Technology, 2025, 437, pp.133068. 10.1016/j.biortech.2025.133068. hal-05201711

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<figure><img src="image_1.png" /></figure>
# Ammonia inhibition in anaerobic digestion and weakly electrical stimulation strategies: Exploring mitigation mechanisms and economic benefits-A review

Jiao Li<sup>a</sup>, Yuchen Zhang<sup>a</sup>, Ruixiang Zou<sup>a</sup>, Wenhui Sun<sup>a</sup>, Wenhui An<sup>a</sup>, Eric Lichtfouse<sup>b</sup>, Suyun Xu<sup>a</sup>, Hongbo Liu<sup>a,*</sup>

## HIGHLIGHTS

* MEC driven microbial metabolism in high ammonia AD are systematically reviewed.

* A systematic description of the mechanism of MEC disinhibition of high ammonia AD.

* Environmental and economic benefits of MEC-AD first to systematically discuss by LCA.

## GRAPHICAL ABSTRACT

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Keywords:**

Anaerobic digestion

Ammonia inhibition

Microbial electrolysis cell

Microbial metabolism mechanisms

Life cycle assessment

## ABSTRACT

Anaerobic digestion (AD) is widely used in waste treatment to produce clean energy such as biogas. However, the excessive ammonia produced during the AD process has a significant inhibitory effect on the microbial system. At present, strategies for alleviating ammonia inhibition have been studied. Among them, microbial electrolysis cell (MEC) emerged as a promising and effective approach in recent years. However, there is still a lack of systematic summary and comprehensive analysis of the mitigation mechanism. Here, ammonia inhibition mechanisms, mitigation mechanisms of MEC and the regulation of microorganisms were comprehensively reviewed. The results show that high ammonia MEC-AD system enhanced the expression of key proteins in hydrogenotrophic methanogens, and methane production shifted from acetoclastic pathway to the hydrogenotrophic pathway. $NH_4^+$ is primarily oxidized at the anode through electrode-mediated pathways and microbial cooperation, enhancing anammox and denitrification gene abundance, promoting $NH_4^+$ and $NO_2$ conversion to nitrogen, thereby alleviating ammonia inhibition. This article provides a comparative assessment of MEC-AD and other technologies from the perspectives of technology, environmental sustainability, and economic feasibility. Supported by life cycle assessment, the MEC-AD system exhibits notable advantages in environmental and economic

<footnote><sup>a</sup> School of Environment and Architecture, University of Shanghai for Science and Technology, 516 Jungong Road, 200093 Shanghai, China</footnote>

<footnote><sup>b</sup> State Key Laboratory of Multiphase Flow in Power Engineering, Xi'an Jiaotong University, Xi'an, Shanxi 710049, China</footnote>
sustainability. However, mechanisms regulating substance conversion and enzyme activity are still unclear in MEC-AD system. In particular, the relationship between active functional microorganisms and their gene expression profiles. Overall, this paper presents the first systematic review of microbial metabolic regulation in high ammonia MEC-AD systems, providing a valuable framework for elucidating the mechanisms of MEC-AD technology alleviates ammonia inhibition.

Abbreviations: AD, anaerobic digestion; FAN, free ammonia; VFA, volatile fatty acids; IET, interspecies electron transfer; EPS, extracellular polymeric substance; LCA, life cycle assessment; LCC, life cycle cost; AK, acetate kinase; AmtB, ammonium transfer protein; EAM, electroactive microorganisms; GHGs, greenhouse gas; MF, magnetic field-assistance; AS, air stripping; TAN, total ammonia nitrogen; CH₄, methane; EET, extracellular electron transfer; DIET, direct interspecies electron transfer; C/N, carbon/nitrogen; MEC, microbial electrolysis cell; HM, hydrogenotrophic methanogenesis; H₄MPT, tetrahydromethotrexate; ETS, electron transport system; AS, air stripping; SCOD, soluble chemical oxygen demand; CCC-MF, current-carrying-coil-based magnetic field.

# 1. Introduction

Anaerobic digestion (AD) is widely considered as a promising technology with low cost and high efficiency (Yuan et al., 2020). As a complex biochemical process, AD depends on synergistic microbial interactions to maintain system stability and ensure effective methanogenesis. However, total ammonia nitrogen (TAN), including NH₄⁺ and free ammonia (FAN) released from protein-containing substrates such as livestock manure, food and other high ammonia wastes, can disrupt this balance (Li et al., 2023).

High ammonia concentrations significantly impair substrate degradation and biogas production in AD, especially in the methanogenic phase (Sun et al., 2024). TAN thresholds range from 1.7 to 14 g/L, with concentrations above 3.4–5.77 g/L reducing methane (CH₄) yields by 39–100% (Yang et al., 2019). Methanogenic archaea are more sensitive to ammonia than hydrolytic and fermentative bacteria. To prevent the accumulation of intermediates such as volatile fatty acids (VFA), they often form syntrophic partnerships with acetogenic bacteria (Yan et al., 2020). Treatment of nitrogen-rich organic wastes can result in high ammonia releases, leading to unbalances in AD process (Bucci et al., 2023). Therefore, effective strategies are urgently needed to enhance the tolerance of microbial to high ammonia concentrations and ensure stable energy recovery from nitrogen-rich wastes.

Various strategies have been employed to mitigate ammonia inhibition in AD, including pH adjustment, increasing the carbon-to-nitrogen (C/N) ratio, substrate dilution and external stimulation technology (Jiang et al., 2019). Although these strategies alleviate ammonia stress in AD, they often increase digestate volume, posing complications for downstream processing. Another strategy involves enhancing ammonia tolerance by domesticating methanogens. However, this method is constrained by the intrinsic ammonia tolerance of the strains. None of these methods fundamentally change the challenges of high ammonia AD. In recent years, coupling electrochemical and AD has shown promise in enhancing microbial stability, improving substrate degradation, and increasing energy conversion efficiency (Wang et al., 2023).

Electrical stimulation has a certain selective effect on the enrichment of microorganisms in AD. Hydrolytic acidogenic bacteria with extracellular electron transfer (EET) capacity are enriched at the anode, promoting organic matter degradation. The lower redox potential at the cathode enables the enrichment of hydrogen-type methanogens and electro-active bacteria. This increases the proportion of methanogens and promotes the cooperation of microbial communities, facilitating interspecies electron transfer (IET) (Sun et al., 2020). Electrochemical stimulation also promotes microorganisms capable of direct interspecies electron transfer (DIET) capable (such as Geobacter), extracellular polymeric substance (EPS), and gene expression of DIET-related proteins (Chen et al., 2023b). High ammonia can promote microorganisms with more stable EPS structures, enhance the self-protection ability, and promote the material and energy exchange to resist ammonia invasion (Zhao et al., 2022).

DIET activation may not be the only mechanism by which external

voltage mitigates ammonia stress in AD. Applied voltage changes in the abundance of nitrogen metabolism genes (anammox, amoA, denitrifying, nirS and nirK) and promote denitrification reactions at the bio-cathode (Chen et al., 2023a; Huang et al., 2022). Thus, electrical stimulation can alleviate ammonia inhibition in AD through symbiotic cooperation with microorganisms and physicochemical pathways. However, the microscopic mechanisms of weakly electrical stimulation to alleviate ammonia inhibition in microbial community studies are currently unknown, especially the effects on ammonia metabolic pathways.

In this paper, a total of 11 articles on ammonia inhibition mitigation strategies were screened and compared, with the screening process and conceptual framework detailed in the Supplementary Materials.

MEC-AD system as an emerging system has been extensively studied in enhancing CH₄ production. The review of the mechanism of ammonia inhibition mitigation using electrochemical methods is still not systematic, and most of the review papers only summarize and compare the strategies of mitigating ammonia inhibition. There is a gap in the review that summarizes MEC technology in comparison to other mitigation strategies for high ammonia suppression both LCA and life cycle cost (LCC) dimensions. Therefore, the most important objectives of this paper are to investigate 1) inhibition mechanism of ammonia on methanogens during AD; 2) the advantages of weakly electrical stimulation to alleviate ammonia inhibition; 3) the metabolic mechanisms of microorganisms in high ammonia MEC-AD systems; and 4) the environmental benefits and economic viability of the MEC-driven AD system from the LCA perspective. This paper aims to provide a sustainability of the MEC-AD application to provide a comprehensive evaluation.

# 2. Transformation of ammonia and toxic effects

Nitrogen is an essential nutrient for microbial growth, and ammonia nitrogen promotes microbial growth at low concentrations (Morales-Polo et al., 2018). In general, anaerobic microorganisms exhibit optimal activity at ammonia concentrations of 50–200 mg/L, remain tolerant at 200–1000 mg/L, but experience inhibition at 1500–3000 mg/L, potentially causing process collapse (Yang et al., 2024). Therefore, this review focuses on the transformation pathways of ammonia in AD and the related molecular mechanisms.

Both NH₄⁺ and FAN directly or indirectly inhibit AD, and the relationship between the two is defined by Eq. 1. FAN is generally regarded as the primary cause of ammonia inhibition. Its concentration is determined by multiple factors, including temperature, organic loading, pH, NH₄⁺ levels, and microbial acclimation, as shown in Eq. 2.

$$NH_4^+ \rightleftharpoons NH_3 + H^+ \quad (1)$$

$$\text{FAN} = \text{TAN} \left( 1 + \frac{10^{-pH}}{10^{-(0.09018 + \frac{272.92}{T})}} \right)^{-1} \quad (2)$$

Where FAN = Free Ammonia Nitrogen Concentration (mg/L), TAN =
Total Ammonia Nitrogen Concentration (mg/L), T = Temperature (K).

The inhibitory mechanisms of ammonia can be categorized into three primary aspects, as illustrated in Fig. 1. Firstly, FAN passively diffuses across the cell membrane and combines with intracellular H⁺ to form NH₄⁺, disrupting intracellular pH homeostasis (Astals et al., 2022). Secondly, the accumulation of NH₄⁺, causes proton imbalance, prompting microbial cells to actively expel potassium ions (K⁺) to maintain pH stability. This compensatory mechanism substantially depletes cellular energy reserves, consequently limiting the availability of ATP for essential enzymatic reactions and cellular metabolism (Mlinar et al., 2022; Peng et al., 2023). Methanospirillum hangatei loses 98% of its intracellular K⁺ at high ammonia concentrations (Sprott et al., 1984). Additionally, high NH₄⁺ concentrations (TAN > 2000 mg/L) inhibit methanogenesis by interfering with methane synthase activity, thereby reducing AD efficiency (Astals et al., 2018).

Ammonia stress exerts a significant inhibitory effect on proteolytic processes. During the acidogenic phase, pyruvate undergoes enzymatic reduction to lactate catalyzed by lactate dehydrogenase. The resulting lactate is then metabolized through the acrylate pathway, where it is converted to propionate by the catalytic action of propionate coenzyme A transferase. Propionate is characterized as difficult to degrade and its accumulation can adversely affect the AD process. This metabolic imbalance subsequently impedes the bioconversion of pyruvate to acetate, ultimately resulting in the suppression of the methanogenic pathway (Li et al., 2020). The inhibition of methanogenic processes triggers a cascade of metabolic disturbances, characterized by the progressive accumulation of VFAs and intermediate metabolites, including propionic acid, butyric acid, and short-chain fatty acids (Tian et al., 2018). Notably, even under ammonia-inhibited conditions, sustained CH₄ production is maintained within the system due to the predominance of methanogens, which exhibit remarkable resistance to ammonia toxicity (Yu et al., 2020). These hydrogenotrophic methanogens preferentially utilize the hydrogenotrophic methanogenesis (HM) pathway, a state known as inhibited steady state (Liu et al., 2025).

In summary, high ammonia concentrations impair AD stability and CH₄ production by disrupting microbial integrity, inhibiting key enzymes, and altering community composition. Various physical, chemical, and biological approaches have been developed to alleviate

ammonia inhibition. However, those often face limitations regarding cost-effectiveness, sustainability, and environmental compatibility. In recent years, MEC has emerged as a promising approach, offering precise control, environmental compatibility, and direct regulation of microbial activity. This strategy provides a new research direction and technical pathway for enhancing AD performance under high ammonia stress.

### 3. Regulation mechanism of high ammonia AD systems by MEC

The MEC provides a low voltage to drive the bioelectrochemical reaction, in which the exoelectrogenic bacteria decompose organic matter at the anode through EET (Eq. 3). These electrons are transferred through an external circuit to the cathode, where they participate in reduction reactions that generate CH₄ (Eq. 4) and H₂ (Eq. 5) (Vrieze et al., 2018).

$$ \text{Anode: } C_2H_4O_2 + 2H_2O \rightarrow 2CO_2 + 8H^+ + 8e^- \quad (3) $$

$$ \text{Cathode: } CO_2 + 8H^+ + 8e^- \rightarrow CH_4 + 2H_2O \quad (4) $$

$$ 2H^+ + 2e^- + 8H^+ \rightarrow H_2 \quad (5) $$

Given the complexity of MEC-AD systems under high ammonia conditions, understanding the influence of weakly electrical stimulation on CH₄ production pathways is crucial for elucidating the mechanisms behind variations in CH₄ yield.

#### 3.1. Effect on microbial community structure

MEC-AD enhances high ammonia AD by regulating microbial community structure. Under an ammonia concentration of 1000 mg/L, applying 0.7 V to the MEC-AD system resulted in a CH₄ content of 20–30 % after 42 days, while the control group showed near-complete functional collapse (Zhang et al., 2022a; Zhang et al., 2022b). A similar conclusion was reported by Cerrillo et al. (2017). Comparative analysis reveals distinct population dynamics between AD and MEC-AD. Desulfovomadaceae emerges as the predominant family under MEC-AD reactors, while Methanobacteriaceae dominates archaeal communities in

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Mechanism of inhibition of AD process by ammonia nitrogen. Ammonia enters the cells of methanogens by diffusion, competes for H⁺, expels K⁺, and fails to maintain ionic balance. The NH₄⁺ formed interferes with enzyme activity and reduces CH₄ production. Acronyms: VFA: volatile fatty acids, TCA: tricarboxylic acid cycle, ATP: adenosine triphosphate.</figcaption></figure>
AD under ammonia stress (Cerrillo et al., 2017). Electrochemical stimulation significantly enriches ammonia-tolerant taxa, with *Desulfovibrio* spp. and *Trichoderma sphaericornutum* populations increasing by 27.4 % and 2.5–6.7 % respectively, correlating with improved VFA degradation capacity (Cao et al., 2022). The key bacteria and archaea involved in MEC-AD are listed in Table 1.

In contrast, weakly electrical stimulation in high ammonia system leads to distinct microbial effects. Under high ammonia conditions (5000 mg/L), *Methanobacterium* exhibited a higher relative abundance compared to *Methanosaeta*. Notably, *Methanobacterium* is a hydrogenotrophic methanogen that converts H₂ and CO₂ into CH₄, whereas *Methanosaeta* is an acetoclastic methanogen that produces CH₄ directly from acetate (Shao et al., 2023). Under elevated ammonia levels, HM pathway becomes thermodynamically more favorable than the acetoclastic pathway as confirmed by standard Gibbs free energy calculations (Eq. 6 and 7) (Pan et al., 2021). This suggests that hydrogenotrophic methanogens can efficiently utilize cathodically produced H₂ or acquire electrons by DIET for CO₂ reduction (Xu et al., 2020). Voltage application regulates H₂ concentration by electrochemical hydrogen evolution, lowers redox potential, and enhances the thermodynamic advantage of HM pathway (Yu et al., 2022). Cathode as an electron donor to generate H₂ or promote the DIET process. It accelerates DIET between hydrogen-producing and methanogenic microorganisms without reliance on intermediate carriers (H₂ or formate) (Xu et al., 2020). Therefore, the HM is the dominant methanogenic pathway under electrical stimulation and high ammonia environments.

$$ \text{Acetate} + 2\text{H}_2\text{O} \rightarrow 2\text{CO}_2 + 4\text{H}_2\Delta\text{G}^\circ(\text{kJ}) = +104.6 \quad (6) $$

$$ \text{CO}_2 + 4\text{H}_2 \rightarrow \text{CH}_4 + 2\text{H}_2\text{O}\Delta\text{G}^\circ(\text{kJ}) = -135.0 \quad (7) $$

In addition, the relative abundance of *Thermovirga* has significantly increased. This genus is involved in the degradation of nitrogen-rich organics, including proteins and amino acids, producing intermediate metabolites like acetate and H₂. These metabolites act as substrates for syntrophic acetate oxidation and HM, indicating a potential syntrophic relationship between *Thermovirga* and methanogens under ammonia stress (He et al., 2020). Based on species-level analysis of archaea, Gram-

<table><caption>**Table 1**
The main bacteria and archaea involved in the MEC-AD process.</caption><thead><tr><th>Phylum</th><th>Genus</th><th>Function</th><th>Ref.</th></tr></thead><tbody><tr><td>Proteobacteria</td><td>Geobacter, Shewanella, Pseudomonas, Desulfovibrio, Desulfuromonas, Sulfurospirillum Clostridium, Syntrophomonas, Enterococcus, Acetobacterium, Streptococcus, Tepidanaerobacter, Caloramator, Coprothermobacter</td><td>Certain bacteria exhibit electroactivity during key anodic or anabolic electron transfer processes. Electroactive, fermentative, and anabolic bacteria cooperate with methanogens to adapt to high ammonia environments.</td><td>Darrer et al., 2023; Yan et al., 2023; Di et al., 2022; Walker et al., 2009; Zhao et al., 2018</td></tr><tr><td>Euryarchaeota</td><td>Methanosarcina, Methanothrix (Methanosaeta), Methanoculleus, Methanomassiliicoccus</td><td>Electroactive archaea involved in methanogenesis by DIET or hydrogenotrophic pathways partially exhibit high ammonia tolerance.</td><td>Kato et al., 2012; Rotaru et al., 2014</td></tr><tr><td>Bacteroidetes</td><td>Parabacteroides, Bacteroides</td><td>Anaerobic degrading bacteria, some with electroactive potential, contribute to the initial breakdown of substrates.</td><td>Zhu et al., 2024</td></tr></tbody></table>

negative or Gram-variable bacteria typically dominate in AD. While Gram-positive species tend to be enriched in MEC-AD. Gram-positive bacteria possess a thick peptidoglycan-based cell wall (20–80 nm) with a stable three-dimensional structure, providing enhanced protection against external penetration (Beom et al., 2018).

Furthermore, MEC-AD stimulates the enrichment and growth of electroactive microorganisms. *Chloromonas tricolor*, an electroproducing microorganism, has been shown to promote H₂ production and enhances CH₄ producing fluxes through acetate conversion (Shao et al., 2019). *Clostridium difficile*, an exoelectrogenic bacterium that converts organic matter to acetate. Increased the ratio of exoelectrogenic bacteria in the MEC-AD system by 10 % compared to conventional AD (Park et al., 2018). In the ammonia perturbation system, the top ten microorganisms in the MEC-AD interacted more closely, resulting in better CH₄ production performance. However, voltage may inhibit anaerobic microbes, leading to a 37.1 % decrease in CH₄ production (Ding et al., 2016). Similarly, Zhang et al. (2021) found that applied voltages of 0.4 and 0.6 V reduced CH₄ production, possibly due to insufficient ammonia for microbial growth.

## 3.2. Regulatory mechanisms of key driver enzymes

As biocatalytic drivers of organic matter degradation, microbial enzymes fundamentally determine AD efficiency. Integrated metagenomic and metaproteomic analyses in high-ammonia systems revealed substantial downregulation (>80 % average reduction) of critical enzymes governing the metabolism of hydrogen and acetic acid production, including acetyl-CoA synthase, methylmalonyl-CoA mutase, succinyl-CoA synthetase, pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, pyruvate oxidoreductase, acetyl-CoA C-acetyltransferase, and acetaldehyde dehydrogenase (Liu et al., 2021). These enzymes are critical for converting propionic and butyric acids to acetic acid. Significantly, methyl-coenzyme M reductase activity in Methanosarcina *spp.* demonstrates concentration-dependent inhibition under endogenous ammonia accumulation, impairing acetoclastic methanogenesis (Zhang et al., 2022a; Zhang et al., 2022b).

In addition, the microbial community adapts to acetate and VFA accumulation by enriching ammonia-tolerant methanogens and upregulating key enzymes involved in acetoclastic methanogenesis, thereby restoring AD performance. Acetic acid and CH₄ production are influenced by microbial secretagogues, including acetate kinase (AK) and F420. AK is essential in acetogenesis, whereas F420 mediates electron transfer during methanogenesis and is considered an important indicator of methanogens activity (Yang et al., 2021). In ammonia inhibited MEC-AD, AK activity was significantly higher than in the control, suggesting that electrical stimulation promotes acetic acid production and increases methanogenic activity in AD (Zhao et al., 2022).

The methanogenic pathway in ammonia stress is primarily hydrogenotrophic, and appropriately applied voltages can stimulate the enzymatic activity of HM. The expression of key intracellular proteins of HM such as coenzyme F420 hydrogenase subunit alpha, 5,10-methylentetrahydromethanopterin reductase, coenzyme F420 hydrogenase subunit gamma, CO dehydrogenase/acetyl-CoA synthase complex subunit epsilon, tetrahydromethanopterin S-methyltransferase subunit H and catalase-peroxidase, was significantly upregulated (Liu et al., 2025). These changes may be crucial for HM in successfully converting H₂/CO₂ to CH₄ under high ammonia stress.

In AD, microbial interspecies electron transfer mainly conducted by H₂ and formate as intermediates. DIET enables direct electron transfer by conductive pili or Cytochrome C, bypassing intermediates like hydrogen or formate. Besides H₂ and formate, applied voltages, external electrodes, and conductive materials in MEC-AD systems provide additional electron donors and pathways to DIET. Cytochrome C was increased 2.4-fold in the MEC-AD system, greatly facilitating the DIET process (Tang et al., 2021). As shown in Fig. 2a, the possible direct electron transport mechanism and enzymes involved in MEC-AD HM in
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Electron transfer and nitrogen metabolism mechanisms in high ammonia MEC-AD systems. (a) Possible direct electron transport mechanisms and enzymes of MEC-AD HM in high ammonia system. Abbreviations: CoM, coenzyme M; CoB, coenzyme B; Fdox, ferredoxin; CoM—S—S—CoB, coenzyme M S—S—coenzyme B; F₄₂₀, low-potential redox cofactor; H₄MPT, tetrahydromethanepterin. a-i represent each stage of the process. Modified from Zhao et al., 2021; (b) The electron transport system and the three electron transport shift pathways of AD; (c) The nitrogen metabolism pathways in the high-ammonia MEC-AD system. Colors represent differences in gene abundance: purple indicates abundance < 10,000; pink, 10,000–20,000; green > 20,000–30,000; blue > 30,000; and no color indicates not detected (ND).</figcaption></figure>

Adapted from Chen et al., 2023a.

a high ammonia system are described (Zhao et al., 2021). In conven-
tional AD, H₂ serves as the sole electron carrier between cells. In MEC-
AD, 1) electrons directly participate in the CO₂ and furan CH₄ connec-
tion, forming CHO-MF, avoiding the hydrogenase-mediated ferrdodoxin
(Fdox, E = −398 mV) reduction process; 2) replaced H₂ as the donor for
formylation of tetrahydromethotrexate (H₄MPT), circumventing the
dual electron requirement of F₄₂₀ reduction (E = 357 mV); 3) the
reduction of CoM—S—S—CoB is catalyzed by isodisulfide reductase,
leading CH₄ production by methyltransferase.

Several proteins associated with reduced ammonia toxicity were
upregulated. The decreased abundance of ammonium transfer protein
(AmtB) suggests reduced ammonia into microbial cells, alleviating
intracellular ammonia toxicity (Nygaard et al., 2006). Additionally,
proteins associated with ammonium detoxification, including glutamine
synthetase and glutamate dehydrogenase, are upregulated, suggesting
that ammonia can be converted to amino acids more rapidly to reduce
toxicity (Song et al., 2022).

Although previous studies have investigated system performance
and microbial community dynamics in AD and MEC-AD under ammonia
stress, the enzymatic response mechanisms in MEC-AD remain insuffi-
ciently characterized. Future studies should assess in depth the potential
active functional microorganisms and their gene expression profiles in
the endogenous ammonia-induced MEC-AD.

3.3. Electron transfer mechanisms

CH₄ production in the system is closely related to IET (microbe-
microbe) and DIET (microbe-solid-state electron acceptor/donor,
referring to electrodes) (Fig. 2b). Although hydrogenotrophic metha-
nogens predominantly depend on H₂ as an electron donor, certain spe-
cies have demonstrated the capability to acquire electrons from the
cathode under electrochemical stimulation (Lee et al., 2018). The un-
derlying mechanisms may involve membrane-associated hydrogenases,
iron-containing redox proteins, or multi-heme cytochromes that facili-
tate direct or mediated electron uptake from the cathode (Xu et al.,
2020). Transmembrane electron carriers facilitate the transfer of elec-
trons across the cell membrane, enabling their entry into intracellular
metabolic pathways for the reduction of CO₂ to CH₄ (Lovley, 2017).

During the DIET process, electrons are initially transferred from the
electrode to the outer membrane of electroactive microorganisms, then
transported across the membrane by conductive pili, Cytochrome C, and
porin-cytochrome complexes. Depend on the electron transport system
(ETS), including complexes of NADH-monoquinone oxidoreductase,
succinate dehydrogenase, cytochrome bc1 and Cytochrome C oxidase,
delivers electrons into the cytoplasm (Anand et al., 2022). In addition,
electrons can be gradually transferred to the cell membrane by succinate
dehydrogenase, cytochrome bc1 and Cytochrome C oxidase. Abundance
of these complexes was upregulated, indicating that the application of voltage promotes electron transfer (Li et al., 2024b). Weakly electrical stimulation enhances EET and transmembrane electron uptake, supporting CH₄ synthesis.

EPS plays a crucial role in microbial electron exchange. Electroinert EPS, such as polysaccharides form the backbone of biofilms, while electroactive EPS including cytochromes and humic acids mediates electron transfer (Yu and Zhang, 2019). Under high ammonia conditions, degradation of inert EPS provides more electroactive sites, facilitating IET between electroactive microorganisms (EAM) and methanogens (Yu and Zhang, 2019). The upregulation of EET related proteins accelerated DIET between EAM and methanogens (Yu et al., 2018a). EPS-derived redox mediators such as quinones, riboflavin, and redox-active proteins promote electron transfer to acceptor microbes (Wang et al., 2022a). Enhanced EPS facilitates syntrophic cooperation and DIET, thereby promoting CH₄ production.

Conductive materials can act as mediators for electron transfer, promoting the co-trophic metabolism between EAM and methanogens (Liu et al., 2012). Conductive materials further facilitate electron transfer by serving as electron shuttles and inducing expression of genes encoding conductive pili, Cytochrome C, and F420 hydrogenase (Liu et al., 2012; Lei et al., 2019). Furthermore, humic acid and redox mediators such as cystine/cysteine facilitate electron shuttling, alleviates ammonia inhibition (Wang et al., 2020).

Voltage application significantly enhanced the abundance and activity of microorganisms related to nitrogen metabolism, improving TAN removal. Voltage stimulation promotes DIET among microorganisms, enhances metabolic activity, and accelerates ammonia conversion. MEC-AD accelerates degradation and CH₄ production by enhancing the hydrolysis and acidification. Meanwhile, increased *mcrA* expression in cathodic methanogens further promotes methanogenesis (Chen et al., 2023a). An anode potential of -0.5 V (vs Ag/AgCl) promotes NH₄⁺ oxidation to NO₂⁻, increases nitrogen removal by 12.3%-29.2%, and significantly enriches anammox bacteria (Zhu et al., 2016). However, studies have reported that at 0.1 g/L of TAN concentration, CH₄ production rate in MEC-AD systems did not improve, possibly due to variations in substrate properties (Zhao et al., 2022).

MEC-AD promotes the abundance of nitrogen-metabolizing microorganisms and the expression of functional genes for anammox and denitrification (Chen et al., 2023a) (Fig. 2c). qPCR results revealed that the abundance of the *amoA* increased by over 20% in MEC-AD, indicating enhanced anammox and promoting the conversion of NH₄⁻-N and NO₂⁻-N to N₂ (Hou et al., 2023). Furthermore, the abundance of key genes for heterogeneous nitrate reduction (*narGHI*) and denitrification (*nirS* and *nirK*) was significantly increased. This enhancement promoted the conversion of NO₂⁻-N to N₂ (Zhang et al., 2019a; Zhang et al., 2019b). Undoubtedly, voltage application boosts the abundance of key genes involved in nitrogen metabolism in the AD, especially denitrification and anammox processes, mitigating ammonia inhibition. However, the low expression level of the *amoA* remains the fundamental constraint to fully overcoming ammonia inhibition (Fig. 2c).

# 4. Application effect evaluation and economic benefit analysis

## 4.1. Comparison of strategies for alleviating ammonia inhibition

Compared to high voltage treatments (>1.5 V), weakly electrical stimulation (0.2–1.2 V) reduces energy consumption and avoids toxicity (Park et al., 2018; Wang et al., 2022b). The technology offers several advantages: 1) the primary by-product is molecular nitrogen (N₂); 2) regulation by voltage to achieve control of ammonia removal; 3) organic matter degradation and energy recovery, enhancing environmental sustainability. At 0.4 V, simultaneous denitrification and AD of Taihu blue algae increased CH₄ yield by 218% (Zheng et al., 2021). Increasing voltage from 0.5-1 V significantly enhanced soluble chemical oxygen demand (SCOD) and VFA degradation (Yu et al., 2018b).

Besides electrical stimulation, other external stimulation techniques such as magnetic field-assistance (MF), light-assisted, and ultrasonic-assistance have been applied to AD. Current-carrying-coil-based magnetic field (CCC-MF) increased CH₄ production by 18.7% and intensified key methanogenic pathways by 9–20 times (Zhang et al., 2024). Static and dynamic MF enhanced CH₄ content by 65.02% and facilitated the formation of struvite (Yang et al., 2023). Nevertheless, the unpredictability of microbial responses constrains to its application in AD. Light-assisted AD also significantly increases CH₄ production by 200–204%, and alleviates ammonia inhibition (Zhu et al., 2022). However, prolonged exposure may reduce methanogenic activity. Studies on the mechanisms of MF or light in mitigating ammonia inhibition remain limited. Ultrasonic-assisted treatment commonly used as a pretreatment technique. Ultrasonic pretreatment increased the SCOD of manure by 41%, raised carbohydrate content by 3.1-fold, and enhanced CH₄ production (Somers et al., 2021). However, its high energy consumption can cause a negative energy balance, limiting economic efficiency.

Other ammonia inhibition mitigation strategies have significant limitations compared to electrochemical techniques. Struvite precipitation enables ammonia recovery while producing valuable fertilizer. However, its precipitation efficiency is regulated by pH, Mg²⁺/PO₄³⁻ molar ratio and residual phosphates tend to cause eutrophication (Tansel et al., 2018). Biological enhancement is rapid, stable, adaptable, and environmentally friendly. An increase of 28% in CH₄ production by introducing ammonia tolerant consortia, but effectiveness drops by 60–80% when TAN > 4000 mg/L, risking microbial imbalance (Tian et al., 2019). C/N ratio adjustment improves ammonia assimilation and waste synergy, but excess carbon (C/N > 30) leads to VFA accumulation and acidification. (Rajagopal et al., 2013). Although membrane separation offers environmental benefits, hollow fiber membranes remove only up to 60% of ammonia in chicken manure AD, insufficient for treating high ammonia wastewater (TAN > 5000 mg/L) (Bayrakdar et al., 2018). Air stripping (AS) is easy to implement and enables recoveries nitrogen as inorganic fertilizers with up to 98% removal efficiency. However, its high energy consumption (2.5–3.5 kWh/m³) limits cost-efficiency versus electrochemical methods (0.8–1.2 kWh/m³) (Vaneecckaute et al., 2017).

Recent studies indicate that adding conductive biochar promotes the growth of ammonia-tolerant methanogens and enhances electron transfer in hydrogenotrophic methanogenic (Yan et al., 2021). However, its high cost and risk of secondary pollution, along with potential disruption of microbial communities, limit its application. Micro-nutrient supplementation remains controversial. Co/Ni/Se can raise the ammonia inhibition threshold to 4200 mg/L, but excessive Fe (>500 mg/L) inhibits hydrolase activity (Molaey et al., 2018; Parra-Orobio et al., 2018). Current researches mostly focus on optimizing a single technology and lacks systematic multi-dimensional comparisons. Table 2 provides a comparative evaluation of weakly electrical stimulation and other approaches.

## 4.2. Environmental cost and benefit analysis

LCA emerged as a critical analytical tool for evaluating environmental impacts across product/service life cycles. These methodologies employ a holistic quantification framework to compare functional equivalents from cradle to grave, with significant applications in optimizing biogas production systems (Visentin et al., 2020).

This paper establishes two LCA objectives: 1) comparative analysis of environmental impacts and sustainability between MEC-AD and conventional AD; 2) evaluation of energy recovery potentials. Adopting a cradle to grave framework, the system boundary initiates at waste collection, encompassing key functional unit operations: material collection, transportation, pretreatment, bioconversion, biogas valorization, and effluent management. System inputs include matter and energy, and outputs include energy recovery and pollutant emissions.
<table><caption>**Table 2**

Comparison of MEC with other mitigating ammonia inhibition techniques.</caption><thead><tr><th>Technology</th><th>Ammonia concentration</th><th>Measure</th><th>COD reduction rate (%)</th><th>VFA removal rate (%)</th><th>TAN removal rate (%)</th><th>CH<sub>4</sub> production rate (%)</th><th>Ref.</th></tr></thead><tbody><tr><td rowspan="3">MEC</td><td>TAN 5000 mg/L</td><td>0.75 V voltage</td><td>75.23</td><td>83.33</td><td>60.84</td><td>78.20</td><td>Zhao et al., 2022</td></tr><tr><td>TAN 6200 mg/L</td><td>0.30 V voltage</td><td>76.10</td><td>54.64</td><td>10.96</td><td>65.90</td><td></td></tr><tr><td>TAN 7000 mg/L</td><td>0.20 V voltage</td><td>/</td><td>/</td><td>20.60</td><td>14.60</td><td></td></tr><tr><td rowspan="2">MF</td><td>/</td><td>CCC-MF, 1.12–3.54 mT</td><td>34.93</td><td>/</td><td>10.40–64.90</td><td>18.70</td><td>Zhang et al., 2024</td></tr><tr><td>/</td><td>Dynamic magnetic field, 10.00–14.00 mT</td><td>/</td><td>/</td><td>/</td><td>65.02</td><td>Yang et al., 2023</td></tr><tr><td rowspan="2">Light-assisted</td><td>TAN 2500 mg/L</td><td>30.00–400.00 W/m<sup>2</sup>, 30 min/d</td><td>/</td><td>/</td><td>37.50</td><td>200.00–204.00</td><td>Zhu et al., 2022</td></tr><tr><td>/</td><td>Solar light</td><td>83.50</td><td>75.40</td><td>60.00</td><td>93.00</td><td>Marín et al., 2022</td></tr><tr><td>Ultrasonic-assistance</td><td>/</td><td>0.20–1.00 W/mL</td><td>62.00–80.00</td><td>/</td><td>/</td><td>40.00</td><td>Joshi and Gogate, 2019</td></tr><tr><td>Struvite precipitation process</td><td>TAN 5790 mg/L</td><td>Add magnesium and phosphorus</td><td>69.48</td><td>/</td><td>50.10</td><td>30.23</td><td>Yang et al., 2024</td></tr><tr><td>Bioaugmentation</td><td>TAN 2000–4900 mg/L</td><td>Non-domesticated mixed microbial consortia</td><td>/</td><td>28.39–59.59</td><td>/</td><td>5.60–13.50</td><td>Li et al., 2024a</td></tr><tr><td>Increase C/N ratio</td><td>TAN 1000–4000 mg/L</td><td>C/N ratio to 2:1</td><td>85.46</td><td>95.48</td><td>94.83</td><td>89.11</td><td>Capson-Tojo et al., 2020</td></tr><tr><td>Membrane separation</td><td>TAN 4500 mg/L</td><td>Vacuum-assisted gas permeable membranes</td><td>/</td><td>/</td><td>80.00</td><td>95.40</td><td>Bayrakdar et al., 2017</td></tr><tr><td rowspan="2">AS</td><td>TAN 6313 mg/kg</td><td>AS with pre-treated air</td><td>34.00</td><td>66.67</td><td>85.00–90.00</td><td>71.79</td><td>Sürmeli et al., 2017; Yao et al., 2017</td></tr><tr><td>TAN 5622 mg/L</td><td>Ammonia aeration of pre-treated air</td><td>89.34</td><td>73.57</td><td>77.00</td><td>62.50</td><td></td></tr><tr><td>Biochar</td><td>TAN 4000 mg/L</td><td>4 g N/L biochar</td><td>62.94</td><td>89.47</td><td>/</td><td>46.00</td><td>Yan et al., 2021</td></tr><tr><td>Conductive materials</td><td>TAN 5500 mg/L</td><td>Addition of zerovalent iron nanoparticles</td><td>75.92</td><td>84.97</td><td>/</td><td>50.60</td><td>Menga et al., 2020</td></tr><tr><td>Trace elements</td><td>TAN 10000 mg/L</td><td>Addition of 1 ml of a mixed trace element</td><td>68.82</td><td>75.78</td><td>/</td><td>70.45</td><td>Wei et al., 2015</td></tr></tbody></table>

The system boundary is shown in Fig. 3.

Inventory analysis involves all material and energy flows associated with AD process, including feedstock acquisition (transportation and pretreatment), energy inputs (water, electricity, gas), and resulting emissions. Studies show MEC offers better environmental sustainability

than MFC and MDC, and biomethane from AD is more beneficial for transport than cogeneration (Ardolino et al., 2018; Zhang et al., 2019a; Zhang et al., 2019b). These findings highlight that MEC offers significant environmental benefits in energy conversion and sustainability. This seems to contradict the research of Amin et al. (2022). Similarly, the

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** System boundary diagram for AD and MEC-AD. Where black arrows represent mainstream processes, red arrows represent emissions in the system, green arrows represent recycling of energy, and blue arrows represent energy inputs to the system.</figcaption></figure>
study also found that the MEC-AD system nearly failed after 80 days, likely due to high OLR inhibiting methanogens (Cerrillo et al., 2016).

Life cycle impact assessment quantifies potential environmental impacts of the inventory stage, including climate change, ecotoxicity, energy consumption, and human toxicity potential, providing a comprehensive analysis for specific environmental factors (Gallego-Ramírez et al., 2023). Studies have shown that lettuce grown in soil amended with AD sludge exhibits higher concentrations of heavy metals (Shamsollahi et al., 2019).

The interpretation stage ultimately assesses the extent to which energy consumption and environmental impacts are reduced throughout the AD process. Several studies have compared the environmental impacts of AD, MEC (0.5 V), and microbial fuel cells (MFC) using the international standard LCA framework show that the MEC offers superior net environmental benefit (Foley et al., 2010). MEC mainly uses electricity but produces valuable by-products like H₂, biodiesel, and H₂O₂, enhancing sustainability and economic benefits (Teo et al., 2023). Table 3 summarizes the environmental sustainability and energy recovery potential of MEC-AD and AD technologies. Notably, studies primarily focus on comparing LCA performance or ammonia inhibition alleviation between MEC-AD and AD. While LCA analyses specifically addressing the role of MEC-AD in mitigating ammonia inhibition remain limited. MEC-AD systems under high ammonia conditions may different from AD in energy consumption patterns, resource flows, and CH₄ production efficiency, highlighting the need for a comprehensive environmental assessment.

Additionally, residual substrates from AD can reduce synthetic nitrogen use by 4.2 kg and cut greenhouse gas (GHGs) emissions (Fernandes et al., 2025). Biogas utilization scenarios (transportation, application) also significantly affect the LAC results. As shown in Fig. 3, under high ammonia, MEC-AD alleviates ammonia inhibition, produces more clean energy, supplies more biogas for energy substitution, emits less GHGs, and is more environmentally sustainable than conventional AD.

### 4.3. Economic cost and benefit analysis

LCC was introduced to assess the economic feasibility of MEC-AD (Li et al., 2024c). Compared to AD, MEC-AD varies in energy use, operation and maintenance, and processing. Thus, cost comparison is essential for evaluating economic sustainability and informing engineering decisions.

The MEC-AD system demonstrates significant economic sustainability. Beegle and Borole (2018) found that while all sludge treatment methods had negative NPVs, MEC-AD had the highest ($22271.82). The goal of the MEC-AD system is to maximize CH₄ production with minimal energy input. In large biogas plants, where the biogas can be used for cogeneration of electricity and heat. The MEC-AD system enhances overall efficiency and generates additional bioenergy, highlighting its potential for practical applications (Wang et al., 2022c).

However, MEC-AD faces several engineering challenges. Precious metal catalysts like platinum improve CH₄ yield but are too costly for large-scale use (Lin et al., 2019). Electrode corrosion and biofilm degradation can impair performance (Hobbsa et al., 2018). Although the MEC-AD system operates at low voltage, precise control of input energy is essential, as excessive voltage can reduce efficiency and induce side reactions (Gao et al., 2020). Additionally, system integration is complex, involving reactor design, membrane selection, and parameter optimization. Membraneless designs reduce costs but risk efficiency loss from substrate diffusion (Amin et al., 2022). Resolving these issues is vital for the advancement of MEC-AD technology.

Cost analysis of MEC-AD under high ammonia conditions remains limited, especially regarding the systematic evaluation for engineering applications. A comprehensive LCC analysis is urgently needed to quantify economic inputs and outputs, providing a theoretical basis and decision support for the optimal design and engineering application of high ammonia AD.

## 5. Future prospects

Although a rich theoretical and research foundation exists for using MEC-AD to mitigate ammonia inhibition, there are still technical

<table><caption>**Table 3**

Environmental and economic benefit cost analysis of traditional AD and MEC-AD.</caption><thead><tr><th>Technology</th><th>Raw materials</th><th>System boundary</th><th>Functional unit</th><th>Input cost (US$)</th><th>GWP (kg CO<sub>2</sub> eq)</th><th>Impact category</th><th>Economic benefit (US$)</th><th>Ref.</th></tr></thead><tbody><tr><td rowspan="4">MEC-AD</td><td>Industrial wastewater</td><td>Reactor construction, AD biological reaction, biogas power generation</td><td>Treat industrial wastewater at 2200 m<sup>3</sup>/d with 4000 mg/L COD</td><td>7.05</td><td>-8.50</td><td>AP, EP</td><td>43.75</td><td>Foley et al., 2010</td></tr><tr><td>Wastewater</td><td>Collection, pretreatment, AD biological process, power generation, and recycling</td><td>The system has a 10-year operational lifespan based on treating 1 L of influent</td><td>66.00</td><td>-8.58</td><td>ETP, EP, HTP</td><td>55.72</td><td>Zhang et al., 2019</td></tr><tr><td>Wastewater</td><td>Collect, transport, process and recycle metallic chromium</td><td>Recover 1 kg chromium metal</td><td>42.37</td><td>-0.44</td><td>ADP, EP, HTP</td><td>254.87</td><td>Muazu et al., 2023</td></tr><tr><td>Wastewater</td><td>Raw material collection, AD biological process, biogas reuse</td><td>Based on the treatment of 1 L wastewater</td><td>0.07</td><td>-7.63</td><td>PMF, HCT, LA, ODP</td><td>0.19-4.99</td><td>Chin et al., 2022</td></tr><tr><td rowspan="4">AD</td><td>Food waste</td><td>Production, packaging, transportation, collection, transportation, incineration or AD</td><td>Manage 1 t food waste for the household</td><td>0.91</td><td>-194.00</td><td>/</td><td>22.56</td><td>Schott and Andersson, 2015</td></tr><tr><td>Garbage</td><td>From waste collection to CH<sub>4</sub> production</td><td>1 kg garbage produces 1 MJ CH<sub>4</sub></td><td>/</td><td>112.00-150.00</td><td>AP, ODP, EP, HTP</td><td>16.71</td><td>Tagliaferri et al., 2016</td></tr><tr><td>Coffee Husk</td><td>Raw material pretreatment, AD, combined heat and power generation, and digestate management</td><td>Process 1 t coffee fruit shells</td><td>9.60</td><td>0.27-0.96</td><td>AP, ETP, EP, HTP</td><td>13.77</td><td>Fernandes et al., 2025</td></tr><tr><td>Wastewater</td><td>Substrate input, AD, biogas output</td><td>Manage 1 t of waste and produce 1 kg of biogas</td><td>1.16</td><td>865.00</td><td>HTP, POCA, HTP, EP</td><td>0.97</td><td>Raketh et al., 2024</td></tr></tbody></table>

Note: GWP stands for Global Warming Potential. The cost of energy input is calculated based on the principle of processing 1 t of waste. Energy input includes water, electricity and transportation. Acronyms: AP: acidification potential, GWP: global warming potential, ETP: ecotoxicity potential, EP: eutrophication potential, HTTP: human toxicity potential, ODP: ozone depletion potential, RI: respiratory inorganics, POCP: photochemical ozone creation potential, and ADP: abiotic depletion potential, fine particulate matter Formation: PMF, Land acidification: LA.
challenges and potential environmental risks in future studies. To better
understand microbial response to ammonia stress in the MEC-AD system
and the mechanisms behind ammonia inhibition behaviors, future
research should focus on the following:

1) Future research could fully integrate macrogenomic, macro-
transcriptomic and macroproteomic data to systematically reveal the
structure and functional characteristics of microbial communities in
the process of high ammonia MEC-AD. This would enable a
comprehensive analysis of the interaction between voltage and mi-
crobial communities, and optimize ammonia mitigation through
gene expression regulation.

2) For the degradation process of dissolved organic matter (DOM) in high-ammonia environments, molecular data from all stages were integrated to explore the potential correlations with microbial gene expression. Further elucidate the specific mechanism of DOM degradation and improve the material transformation pathway.

3) From the perspectives of resource recovery and engineering appli-
cations, modern molecular biology techniques are utilized to identify
microorganisms, especially those capable of CH4 production in high
ammonia environments. We can investigate in depth the mechanism
of their ammonia tolerance and CH4 production. It is conducive to
achieving efficient recovery and resource utilization of CH4.

4) Develop advanced intelligent algorithms to automate the identifi-
cation and analysis of complex genomic features, improving data
analysis efficiency, accuracy and reliability, thereby providing more
precise support for large-scale genomic research.

6. Conclusion

Ammonia inhibition remains a major challenge in AD. This paper reviews the inhibition mechanisms of ammonia and how MEC alleviates inhibition by regulating microbial communities. The results show that ammonia in the AD system reduces CH4 production by disrupting the ion balance and lowering enzyme activity. In high-ammonia MEC-AD, the production of CH4 is metabolically transferred to the HM pathway. Weakly electrical stimulation promotes DIET, enhancing microbial interactions. On the other hand, weakly electrical stimulation enhances the expression of anammox and denitrification genes, promotes the conversion of ammonia to N2, and reduces the toxic effects of ammonia. Furthermore, LCA and LCC analyses indicate that the MEC-AD system offers superior environmental and economic advantages. However, the research on enzyme activity and substrate transformation pathways in high ammonia MEC-AD systems is limited. This is an important direction for future research. This article provides theoretical support for the utilization of nitrogen-rich waste.

CRediT authorship contribution statement

Jiao Li: Writing – original draft, Methodology, Investigation, Formal analysis. Yuchen Zhang: Writing – review & editing, Methodology. Ruixiang Zou: Methodology, Investigation. Wenhui Sun: Methodology, Investigation. Wenhui An: Methodology, Investigation. Eric Lichtfouse: Writing – review & editing. Suyun Xu: Writing – review & editing, Conceptualization. Hongbo Liu: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

**Acknowledgements**

The authors would like to acknowledge the co-funding of this work
by Projects of International Cooperation Shanghai (STCSM,
23230711300) and the National Natural Science Foundation of China
(No. 52470049).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2025.133068.

**Data availability**

Data will be made available on request.

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# Supplementary Materials

1

2 **List of Figures and Tables**

3 **Fig. S1** The conceptual framework of the literature screening process

4 **Table S1** Summarizes and compares the evaluation results

5
In this paper, we used Web of Science database and entered the keywords anaerobic digestion, ammonia inhibition, electrical stimulation, life cycle assessment (LCA), microbial electrolysis cell (MEC) to screen the literature in the last five years (2020-2025), and a total of 587 articles were screened. The conceptual framework of the screening process is shown in Fig. S1. In the second round of review, we systematically analyzed the screened literature to identify key innovations and research gaps in addressing ammonia inhibition in AD. The screening criteria were as follows: comparison of means containing ammonia inhibition by AD; containing electrical stimulation to alleviate ammonia inhibition; LCA analysis of means containing ammonia inhibition, and a total of 11 review papers were screened. The summary and comparison are shown in Table S1. Existing studies primarily focus on the mechanisms of ammonia inhibition mitigation by MEC-AD and the environmental and economic assessments of various technologies, particularly by LCA.

<figure><img src="image_6.png" /><figcaption>Fig. S1 The conceptual framework of the literature screening process</figcaption></figure>
<table><caption>Table S1 Summarizes and compares the evaluation results</caption><thead><tr><td>Highlights</td><td>Gaps in current studies and implications</td><td>Innovations in this review paper</td><td>Ref.</td></tr></thead><tbody><tr><td>Focuses on describing the means of alleviating ammonia inhibition by bioaugmentation strategies and the mechanism of disinhibition.</td><td>Lack of comprehensive economic feasibility analysis of bioaugmentation assisted AD under different scenarios, resulting in limited experimental results.</td><td>A comparative analysis was conducted, focusing on the MEC-AD method for alleviating ammonia inhibition and its economic viability.</td><td>Chen et al., 2025</td></tr><tr><td>Research on the mitigation of ammonia inhibition by bioaugmentation is highlighted, comparing the bioaugmentation process under temperature and reactor configuration.</td><td>Lack of clarity on the economics of bioaugmentation and whether have more advantages compared to other technologies.</td><td>Various technologies were compared and the economic and environmental benefits of MEC-AD were expounded.</td><td>Li et al., 2023</td></tr><tr><td>The recovery of AD system from ammonia stress was discussed, especially H<sub>2</sub> transfer, interspecies electron transfer (MIET) and DIET mechanism.</td><td>There is no comparative summary of the advantages between technologies.</td><td></td><td>Xu et al., 2020</td></tr><tr><td>The molecular mechanisms of ammonia inhibition are outlined, and various mitigation strategies are proposed and compared.</td><td>Lack of environmental and economic benefit comparisons between technologies, no presentation of MEC technologies.</td><td></td><td>Yang et al., 2024</td></tr></tbody></table>
<table>
   <tr>
    <td>Review the studies of nitrogen-rich wastewater as feedstock for AD, and the effects of ammonia inhibition/toxicity on AD are analyzed.</td>
    <td>The mechanisms underlying ammonia inhibition have not been explained, and techniques to alleviate ammonia inhibition are lacking. Technologies comparative analysis of environmental and economic benefits is unclear; comparison of MEC technologies is not available.</td>
    <td>Jiang et al., 2019</td>
   </tr>
   <tr>
    <td>Possible strategies to mitigate ammonia inhibition are described.</td>
    <td>The economic feasibility of the operating conditions of AS needs to be evaluated. Mechanism of AS mitigation of ammonia suppression is currently unclear. Ammonia recovery technologies have not included a discussion of MEC, and the mechanisms of ammonia inhibition are still not summarized.</td>
    <td>Song et al., 2023</td>
   </tr>
   <tr>
    <td>Effective strategies for AS to mitigate high ammonia AD while recovering ammonia from the system are highlighted.</td>
    <td>The technical characteristics of various strategies were compared. The method of alleviating ammonia inhibition by MEC-AD and the mechanisms were described.</td>
    <td>Yellezuome et al., 2022</td>
   </tr>
   <tr>
    <td>The advantages and limitations of AS, membrane separation and struvite precipitation were discussed. Economic analysis of different processes has been conducted.</td>
    <td></td>
    <td>Yang et al., 2022</td>
   </tr>
  </table>
The inhibition effect of ammonia under different conditions and the regulatory measures were compared. In particular, the mechanism of ammonia stress was explained.

Mechanisms for mitigating ammonia inhibition are synthesized from an electron transfer perspective, and several strategies (mainly additives) are proposed. Discussed ammonia inhibition mitigation strategies and the effects of ammonia inhibition on the oxidation of coprophilic propionate (SPO).

Introduce AS technology, especially the recovery and utilization of ammonia and its influence on CH₄ production.

Provides insights into DIET microbial communities, mechanisms, stimulus measures and detection methods in AD.

No discussion of MEC technology or compare the economic and environmental aspects of different approaches.

Lack of environmental and economic benefit comparisons between additives, the role of MEC for electron transfer is not mentioned.

Lack of environmental and economic comparisons between technologies and elaboration on MEC.

AS is not compared with other technologies, and cannot well highlight the advantages in technical and economic environment.

No specific measures to alleviate ammonia inhibition were proposed.

Yang et al., 2024

James et al., 2025

Adams, 2024

Not only were various ammonia mitigation strategies proposed, but technicalities were compared, and the LCA analysis of MEC-AD was further discussed.

Yellezuome et al., 2022

Akram et al., 2024
22

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