CO₂ fixation in anaerobic biological treatment: amorphous carbon formation driven by electron bifurcation

Abstract

Anaerobic digestion (AD) is a commonly used technology for facilitating carbon fixation by converting complex organic matter into volatile fatty acids and CH₄; however, the issue of CO₂ emission remains unresolved in AD. The formation of amorphous carbon has been identified as a more direct method of carbon fixation in AD. This study aimed to elucidate how amorphous carbon can be formed from organic matter or CO₂ by anaerobic microorganisms. The results showed that amorphous carbon was produced in the anaerobic digestion of inorganic and mixed carbon sources, with yields of 0.38 and 3 µg/10⁵ cells, respectively. Its characteristics were analyzed using Raman microscopy. Isotope labeling revealed that CO₂ fixation into amorphous carbon primarily depends on the reversed oxidative tricarboxylic acid cycle (roTCA) and hydroxycaproate. Differential pulse voltammetry combined with gene abundance analysis indicated that flavin electron bifurcation (EB) is involved in electron transfer. The microbial isothermal calorimeter further measured the metabolic calorific value, demonstrating that anaerobic microorganisms can autotrophically fix CO₂ with energy provided by EB. Metagenomic analysis supported the large REDOX equivalents input from EB to sustain the roTCA cycle. This research contributes to understanding the mechanism of CO₂ fixation into solid carbon in anaerobic environments. Additionally, it provides new insights into the potential development of carbon-negative technologies in anaerobic biological treatment.

Graphical abstract

Keywords

Anaerobic digestion / Amorphous carbon / Electron bifurcation / Anaerobic microbial / CO ₂ fixation

Highlight

	● Anaerobic microorganisms immobilize CO2 into amorphous carbon.
	● Electron bifurcation provides energy for CO2 biofixation in the roTCA cycle.
	● Hydroxycaproicate is an intermediate in the formation of amorphous carbon.
	● The REDOX equivalents generated by electron bifurcation support CO2 biofixation.

References

[1] Allen K D, Wegener G, Matthew Sublett D Jr, Bodnar R J, Feng X, Wendt J, White R H. (2021). Biogenic formation of amorphous carbon by anaerobic methanotrophs and select methanogens. Science Advances, 7(44): eabg9739

[2] Buckel W, Thauer R K. (2018). Flavin-based electron bifurcation, a new mechanism of biological energy coupling. Chemical Reviews, 118(7): 3862–3886

[3] Camargo F P, Sakamoto I K, Delforno T P, Midoux C, Duarte I C S, Silva E L, Bize A, Varesche M B A. (2023). Microbial and functional characterization of granulated sludge from full-scale UASB thermophilic reactor applied to sugarcane vinasse treatment. Environmental Technology, 44(21): 3141–3160

[4] Chan H, Babayan V, Blyumin E, Gandhi C, Hak K, Harake D, Liu H Y. (2010). The p-type ATPase superfamily. Journal of Molecular Microbiology and Biotechnology, 19(1−2): 5–104

[5] Coppola A I, Wagner S, Lennartz S T, Seidel M, Ward N D, Dittmar T, Santín C, Jones M W. (2022). The black carbon cycle and its role in the Earth system. Nature Reviews. Earth & Environment, 3(8): 516–532

[6] Corvec S, Seiler E, Wang L, Moreno M G, Trampuz A. (2020). Characterization of medical relevant anaerobic microorganisms by isothermal microcalorimetry. Anaerobe, 66: 102282

[7] Hu W, Peng Y, Wei Y, Yang Y. (2023). Application of electrochemical impedance spectroscopy to degradation and aging research of lithium-ion batteries. Journal of Physical Chemistry C, 127(9): 4465–4495

[8] Jedwab J, Boulègue J. (1984). Graphite crystals in hydrothermal vents. Nature, 310(5972): 41–43

[9] Jia Y, Qian D, Chen Y, Hu Y. (2021). Intra/extracellular electron transfer for aerobic denitrification mediated by in-situ biosynthesis palladium nanoparticles. Water Research, 189: 116612

[10] Kayastha K, Vitt S, Buckel W, Ermler U. (2021). Flavins in the electron bifurcation process. Archives of Biochemistry and Biophysics, 701: 108796

[11] Lubner C E, Jennings D P, Mulder D W, Schut G J, Zadvornyy O A, Hoben J P, Tokmina-Lukaszewska M, Berry L, Nguyen D M, Lipscomb G L. . (2017). Mechanistic insights into energy conservation by flavin-based electron bifurcation. Nature Chemical Biology, 13(6): 655–659

[12] Mackey M C. (1989). The dynamic origin of increasing entropy. Reviews of Modern Physics, 61(4): 981–1015

[13] Mason-Jones K, Breidenbach A, Dyckmans J, Banfield C C, Dippold M A. (2023). Intracellular carbon storage by microorganisms is an overlooked pathway of biomass growth. Nature Communications, 14(1): 2240

[14] Meng F, Han S, Lin L, Li J, Chen K, Jiang J. (2024). Process optimization and mechanism study of ionic liquid-based mixed amine biphasic solvents for CO₂ capture in biogas upgrading procedure. Frontiers of Environmental Science & Engineering, 18(8): 95

[15] Müller V, Chowdhury N P, Basen M. (2018). Electron bifurcation: a long-hidden energy-coupling mechanism. Annual Review of Microbiology, 72(1): 331–353

[16] Nunoura T, Chikaraishi Y, Izaki R, Suwa T, Sato T, Harada T, Mori K, Kato Y, Miyazaki M, Shimamura S. . (2018). A primordial and reversible TCA cycle in a facultatively chemolithoautotrophic thermophile. Science, 359(6375): 559–563

[17] Peters J W, Beratan D N, Bothner B, Dyer R B, Harwood C S, Heiden Z M, Lu Y, Hille R, Jones A K, King P W. . (2018). A new era for electron bifurcation. Current Opinion in Chemical Biology, 47: 32–38

[18] Ragsdale S W. (2018). Stealth reactions driving carbon fixation. Science, 359(6375): 517–518

[19] Santos Correa S, Schultz J, Lauersen K J, Soares Rosado A. (2023). Natural carbon fixation and advances in synthetic engineering for redesigning and creating new fixation pathways. Journal of Advanced Research, 47: 75–92

[20] Steffens L, Pettinato E, Steiner T M, Mall A, König S, Eisenreich W, Berg I A. (2021). High CO₂ levels drive the TCA cycle backwards towards autotrophy. Nature, 592(7856): 784–788

[21] Swuec P, Chaves-Sanjuan A, Camilloni C, Vanoni M A, Bolognesi M. (2019). Cryo-EM structures of Azospirillum brasilense glutamate synthase in its oligomeric assemblies. Journal of Molecular Biology, 431(22): 4523–4526

[22] Thevasundaram K, Gallagher J J, Cherng F, Chang M C Y. (2022). Engineering nonphotosynthetic carbon fixation for production of bioplastics by methanogenic archaea. Proceedings of the National Academy of Sciences of the United States of America, 119(23): e2118638119

[23] Tong Y D, Liao X W, He Y Y, Cui X M, Wishart M, Zhao F, Liao Y L, Zhao Y X, Lv X B, Xie J W. . (2024). Mitigating greenhouse gas emissions from municipal wastewater treatment in China. Environmental Science and Ecotechnology, 20: 100341

[24] Velázquez-CampoyA, OhtakaH, NezamiA, MuzammilS, Freire E (2004). Isothermal titration calorimetry. Current Protocols in Cell Biology, 23(1): 17.18. 11–17.18. 24

[25] Wang Y F, Chen F, Guo H X, Sun P Z, Zhu T T, Horn H, Liu Y W. (2024). Permanganate (PM) pretreatment improves medium-chain fatty acids production from sewage sludge: the role of PM oxidation and in-situ formed manganese dioxide. Water Research, 249: 120869

[26] Yamaoka S, Shaji Kumar M D, Kanda H, Akaishi M. (2002). Crystallization of diamond from CO₂ fluid at high pressure and high temperature. Journal of Crystal Growth, 234(1): 5–8

[27] Yang S, Li S, Jia X. (2019). Production of medium chain length polyhydroxyalkanoate from acetate by engineered Pseudomonas putida KT2440. Journal of Industrial Microbiology & Biotechnology, 46(6): 793–800

[28] Zanello P. (2018). Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part II. Fe₄S₄ and Fe₃S₄ iron-sulfur proteins. Journal of Structural Biology, 202(3): 250–263

[29] Zarzycki J, Brecht V, Müller M, Fuchs G. (2009). Identifying the missing steps of the autotrophic 3-hydroxypropionate CO₂ fixation cycle in Chloroflexus aurantiacus. Proceedings of the National Academy of Sciences of the United States of America, 106(50): 21317–21322

[30] Zhang T Y, Zhang J X, Zhang P S, Wang J B, He Y L. (2023). Electronic bifurcation: a new perspective on Fe bio-utilization in anaerobic digestion of lactate. Environmental Science & Technology, 57(28): 10448–10457

[31] Zheng R, Zhang K, Kong L, Liu S. (2024). Research progress and prospect of low-carbon biological technology for nitrate removal in wastewater treatment. Frontiers of Environmental Science & Engineering, 18(7): 80

[32] Zheng Y, Chen J C, Ma Y M, Chen G Q. (2020). Engineering biosynthesis of polyhydroxyalkanoates (PHA) for diversity and cost reduction. Metabolic Engineering, 58: 82–93

RIGHTS & PERMISSIONS

Higher Education Press 2025