Organic electron donor limitation triggers metabolic trade-offs in anaerobic granular sludge: The pivotal role of Fe-mediated electron bifurcation

Bochuang Huang; Yangyang Li; Jinhao Zhu; Juncong Zou; Dexin Wang; Chunping Yang; Hongjun Han; Wencheng Ma

doi:10.1016/j.cej.2025.172282

Organic electron donor limitation triggers metabolic trade-offs in anaerobic granular sludge: The pivotal role of Fe-mediated electron bifurcation

Bochuang Huang
, Yangyang Li
, Jinhao Zhu
, Juncong Zou
, Dexin Wang^*
, Chunping Yang
, Hongjun Han
, Wencheng Ma

^*Corresponding author for this work

School of Environment

Hainan University
Sanya Central Hospital (Hainan Third People's Hospital)
Guangdong University of Petrochemical Technology
Harbin Institute of Technology

Research output: Contribution to journal › Article › peer-review

Abstract

Fe-dependent electron bifurcation (EB) functions as a unique energy-generating strategy and a key component of metabolic trade-offs that enable methanogens to thrive under organic electron donor-limited environment. However, its molecular mechanism in maintaining the balance between methanogenic activities and morphological stability of sludge remains overlooked. In this study, Fe-based materials including Fe₃O₄, Fe₂O₃, zero-valent iron (ZVI), and FeCl₂ as stimulators were selected to assist with anaerobic granular sludge (AGS) under influent organic-limited condition for identifying EB-based energy-regulating strategy. The results showed that Fe₃O₄-amended AGS with more stable sludge morphology obtained optimum soluble chemical oxygen demand (SCOD) removal efficiency and CH₄ production (88.3 ± 2.5 % and 215.7 ± 8.5 mL/d), which were on average 24.1 % and 56.4 % higher than control and other Fe-treated groups. Furthermore, metagenomic analysis revealed that up-regulated EB-related mvhAGD-hdrABC genes were taxonomically assigned to Methanothrix (Fe₃O₄/Fe₂O₃) and Methanobacterium (ZVI), while those encoding hdrA₂B₂C₂ were affiliated with Methanolinea (FeCl₂). Collectively, these findings indicated that the targeted enrichment of EB-associated methanogens is contingent upon their trade-off decisions to maximize energy metabolism by leveraging Fe bioavailability. More importantly, EB-coupled direct interspecies electron transfer (Fe₃O₄) achieves maximum energy-saving potential (27 kJ/mol) when compared to sole interspecies hydrogen transfer (IHT), sole EB, and EB-coupled IHT, confirming that stress resistance of AGS structure may stem from energy-saving mode switching. This study unveils Fe-mediated EB as an alternative and convenient energy metabolism strategy, which plays a pivotal role in maintaining methanogenic activities and morphological balance under low organic electron donor availability.

Original language	English
Article number	172282
Journal	Chemical Engineering Journal
Volume	528
DOIs	https://doi.org/10.1016/j.cej.2025.172282
State	Published - 15 Jan 2026

Keywords

Anaerobic digestion, anaerobic granular sludge, Electron bifurcation
Energy-regulating strategy
Trade-offs effect

Access to Document

10.1016/j.cej.2025.172282

Cite this

@article{dd12d13fff8542309d8d907019c768f0,

title = "Organic electron donor limitation triggers metabolic trade-offs in anaerobic granular sludge: The pivotal role of Fe-mediated electron bifurcation",

abstract = "Fe-dependent electron bifurcation (EB) functions as a unique energy-generating strategy and a key component of metabolic trade-offs that enable methanogens to thrive under organic electron donor-limited environment. However, its molecular mechanism in maintaining the balance between methanogenic activities and morphological stability of sludge remains overlooked. In this study, Fe-based materials including Fe3O4, Fe2O3, zero-valent iron (ZVI), and FeCl2 as stimulators were selected to assist with anaerobic granular sludge (AGS) under influent organic-limited condition for identifying EB-based energy-regulating strategy. The results showed that Fe3O4-amended AGS with more stable sludge morphology obtained optimum soluble chemical oxygen demand (SCOD) removal efficiency and CH4 production (88.3 ± 2.5 \% and 215.7 ± 8.5 mL/d), which were on average 24.1 \% and 56.4 \% higher than control and other Fe-treated groups. Furthermore, metagenomic analysis revealed that up-regulated EB-related mvhAGD-hdrABC genes were taxonomically assigned to Methanothrix (Fe₃O₄/Fe₂O₃) and Methanobacterium (ZVI), while those encoding hdrA₂B₂C₂ were affiliated with Methanolinea (FeCl₂). Collectively, these findings indicated that the targeted enrichment of EB-associated methanogens is contingent upon their trade-off decisions to maximize energy metabolism by leveraging Fe bioavailability. More importantly, EB-coupled direct interspecies electron transfer (Fe3O4) achieves maximum energy-saving potential (27 kJ/mol) when compared to sole interspecies hydrogen transfer (IHT), sole EB, and EB-coupled IHT, confirming that stress resistance of AGS structure may stem from energy-saving mode switching. This study unveils Fe-mediated EB as an alternative and convenient energy metabolism strategy, which plays a pivotal role in maintaining methanogenic activities and morphological balance under low organic electron donor availability.",

keywords = "Anaerobic digestion, anaerobic granular sludge, Electron bifurcation, Energy-regulating strategy, Trade-offs effect",

author = "Bochuang Huang and Yangyang Li and Jinhao Zhu and Juncong Zou and Dexin Wang and Chunping Yang and Hongjun Han and Wencheng Ma",

note = "Publisher Copyright: {\textcopyright} 2025 Elsevier B.V.",

year = "2026",

month = jan,

day = "15",

doi = "10.1016/j.cej.2025.172282",

language = "英语",

volume = "528",

journal = "Chemical Engineering Journal",

issn = "1385-8947",

publisher = "Elsevier B.V.",

}

TY - JOUR

T1 - Organic electron donor limitation triggers metabolic trade-offs in anaerobic granular sludge

T2 - The pivotal role of Fe-mediated electron bifurcation

AU - Huang, Bochuang

AU - Li, Yangyang

AU - Zhu, Jinhao

AU - Zou, Juncong

AU - Wang, Dexin

AU - Yang, Chunping

AU - Han, Hongjun

AU - Ma, Wencheng

PY - 2026/1/15

Y1 - 2026/1/15

N2 - Fe-dependent electron bifurcation (EB) functions as a unique energy-generating strategy and a key component of metabolic trade-offs that enable methanogens to thrive under organic electron donor-limited environment. However, its molecular mechanism in maintaining the balance between methanogenic activities and morphological stability of sludge remains overlooked. In this study, Fe-based materials including Fe3O4, Fe2O3, zero-valent iron (ZVI), and FeCl2 as stimulators were selected to assist with anaerobic granular sludge (AGS) under influent organic-limited condition for identifying EB-based energy-regulating strategy. The results showed that Fe3O4-amended AGS with more stable sludge morphology obtained optimum soluble chemical oxygen demand (SCOD) removal efficiency and CH4 production (88.3 ± 2.5 % and 215.7 ± 8.5 mL/d), which were on average 24.1 % and 56.4 % higher than control and other Fe-treated groups. Furthermore, metagenomic analysis revealed that up-regulated EB-related mvhAGD-hdrABC genes were taxonomically assigned to Methanothrix (Fe₃O₄/Fe₂O₃) and Methanobacterium (ZVI), while those encoding hdrA₂B₂C₂ were affiliated with Methanolinea (FeCl₂). Collectively, these findings indicated that the targeted enrichment of EB-associated methanogens is contingent upon their trade-off decisions to maximize energy metabolism by leveraging Fe bioavailability. More importantly, EB-coupled direct interspecies electron transfer (Fe3O4) achieves maximum energy-saving potential (27 kJ/mol) when compared to sole interspecies hydrogen transfer (IHT), sole EB, and EB-coupled IHT, confirming that stress resistance of AGS structure may stem from energy-saving mode switching. This study unveils Fe-mediated EB as an alternative and convenient energy metabolism strategy, which plays a pivotal role in maintaining methanogenic activities and morphological balance under low organic electron donor availability.

AB - Fe-dependent electron bifurcation (EB) functions as a unique energy-generating strategy and a key component of metabolic trade-offs that enable methanogens to thrive under organic electron donor-limited environment. However, its molecular mechanism in maintaining the balance between methanogenic activities and morphological stability of sludge remains overlooked. In this study, Fe-based materials including Fe3O4, Fe2O3, zero-valent iron (ZVI), and FeCl2 as stimulators were selected to assist with anaerobic granular sludge (AGS) under influent organic-limited condition for identifying EB-based energy-regulating strategy. The results showed that Fe3O4-amended AGS with more stable sludge morphology obtained optimum soluble chemical oxygen demand (SCOD) removal efficiency and CH4 production (88.3 ± 2.5 % and 215.7 ± 8.5 mL/d), which were on average 24.1 % and 56.4 % higher than control and other Fe-treated groups. Furthermore, metagenomic analysis revealed that up-regulated EB-related mvhAGD-hdrABC genes were taxonomically assigned to Methanothrix (Fe₃O₄/Fe₂O₃) and Methanobacterium (ZVI), while those encoding hdrA₂B₂C₂ were affiliated with Methanolinea (FeCl₂). Collectively, these findings indicated that the targeted enrichment of EB-associated methanogens is contingent upon their trade-off decisions to maximize energy metabolism by leveraging Fe bioavailability. More importantly, EB-coupled direct interspecies electron transfer (Fe3O4) achieves maximum energy-saving potential (27 kJ/mol) when compared to sole interspecies hydrogen transfer (IHT), sole EB, and EB-coupled IHT, confirming that stress resistance of AGS structure may stem from energy-saving mode switching. This study unveils Fe-mediated EB as an alternative and convenient energy metabolism strategy, which plays a pivotal role in maintaining methanogenic activities and morphological balance under low organic electron donor availability.

KW - Anaerobic digestion, anaerobic granular sludge, Electron bifurcation

KW - Energy-regulating strategy

KW - Trade-offs effect

UR - https://www.scopus.com/pages/publications/105027450056

U2 - 10.1016/j.cej.2025.172282

DO - 10.1016/j.cej.2025.172282

M3 - 文章

AN - SCOPUS:105027450056

SN - 1385-8947

VL - 528

JO - Chemical Engineering Journal

JF - Chemical Engineering Journal

M1 - 172282

ER -

Organic electron donor limitation triggers metabolic trade-offs in anaerobic granular sludge: The pivotal role of Fe-mediated electron bifurcation

Abstract

Keywords

Access to Document

Other files and links

Fingerprint

Cite this