Optimizing anaerobic Co-digestion of antibiotic fermentation residue and sludge with iron nanoparticles: Impacts on methane production, microbial dynamics, and biocompatibility

Abstract

The high antibiotic levels in antibiotic fermentation residues (AFR) impede their disposal or resource recovery due to their recalcitrance and toxicity, limiting organic matter degradation and methane production efficiency. This study has investigated the effects of varying-valence iron nanoparticles (Fe NPs) on the anaerobic Co-digestion (Co-AD) process of AFR and waste activated sludge (WAS), focusing on methane production, resistance gene expression, virulence factors, and microbial community interactions. The incorporation of varying-valence Fe NPs enhanced methane yield, with Fe³⁺ NPs increasing it by approximately 1.46-fold, effectively improving the biodegradation of recalcitrant bacterial residue. Fe³⁺ NPs facilitated microbial Fe(III) reduction under anaerobic conditions, thereby supporting enhanced electron transfer, redox activity, and methanogenic metabolism in the Co-AD system as evidenced by a 40.2% increase in cytochrome C. Microbiological analysis revealed that Fe³⁺ NPs upregulate genes associated with methanogenesis (frh, pta, mtr, and fwd), improving electron transfer between Paraclostridium and Methanobacterium. Additionally, Fe NPs induced enrichment of resistance genes and virulence factors, indicating microbial stress from oxidative and membrane perturbations, whereas Fe³⁺ NPs showed markedly weaker effects, superior biocompatibility, and the highest methane production. Fe NPs also promoted microbial metabolic activity and biofilm formation, optimizing Co-AD efficiency. Importantly, Fe³⁺ NPs exhibited better biocompatibility, with lower lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activities. By overcoming the limitations of conventional antibiotic fermentation residue treatment, this study highlights the potential of Fe NPs, particularly Fe³⁺ NPs, in enhancing anaerobic organics bio-methanation and provides new insights into energy-efficient recalcitrant and toxic organic waste treatment strategies.

Graphical abstract

Keywords

Antibiotic fermentation residues / Anaerobic co-digestion / Iron-based nanoparticles / Methanogenesis / Quorum sensing / Resistance and virulence genes

Highlight

	● Fe3+ NPs enhanced methane yield by 46.43% to 145.27 mL CH4/gVS.
	● Fe3+ NPs improved SCOD and TVFAs, enhancing hydrolysis and acidification.
	● Fe3+ NPs enriched Methanobacterium and enhanced DIET-based methanogenesis.
	● Fe3+ NPs showed lowest cytotoxicity and greater microbial compatibility of Fe-NPs.

References

[1] Andronikou M , Christoforou P , Constantinou D , Charalambous P , Samanides C G , Karachaliou P , Vyrides I . (2025). Critical role of bicarbonate in zero-valent iron for hydrogen generation and biogas upgrading in anaerobic digestion. Bioresource Technology, 426: 132236

[2] Aziz A , Sengar A , Basheer F , Farooqi I H , Isa M H . (2022). Anaerobic digestion in the elimination of antibiotics and antibiotic-resistant genes from the environment: a comprehensive review. Journal of Environmental Chemical Engineering, 10(1): 106423

[3] Bennett B D , Gralnick J A . (2019). Mechanisms of toxicity by and resistance to ferrous iron in anaerobic systems. Free Radical Biology and Medicine, 140: 167–171

[4] Carrère H , Dumas C , Battimelli A , Batstone D J , Delgenès J P , Steyer J P , Ferrer I . (2010). Pretreatment methods to improve sludge anaerobic degradability: a review. Journal of Hazardous Materials, 183(1−3): 1–15

[5] Chen C , Hall S , Coward E , Thompson A . (2020). Iron-mediated organic matter decomposition in humid soils can counteract protection. Nature Communications, 11(1): 2255

[6] Cheng Q , Huang W , Jiang M , Xu C , Fan G , Yan J , Chai B , Zhang Y , Zhang Y , Zhang S . et al. (2021). Challenges of anaerobic digestion in China. International Journal of Environmental Science and Technology, 18(11): 3685–3696

[7] Fan X , Zhang g Z , Li N , Li X . (2025). Molecular ecological insights into the synergistic response mechanism of nitrogen transformation, electron flow and antibiotic resistance genes in aerobic activated sludge systems driven by sulfamethoxazole and/or trimethoprim stresses. Water Research, 270: 122853

[8] Feng Y H , Zhang Y B , Quan X , Chen S . (2014). Enhanced anaerobic digestion of waste activated sludge digestion by the addition of zero valent iron. Water Research, 52: 242–250

[9] Fu J W , He Y F , Zhao H X , Yang H , Li Q , Chen R , Li Y Y . (2025). Biochar mediated microbial synergy in Partial nitrification –anammox systems: enhancing nitrogen removal efficiency and stability. Frontiers of Environmental Science & Engineering, 19(7): 95

[10] Fu J W , Hou Z Y , Zhao H X , Li Q , Chen R , Li Y Y . (2024). Enhanced nitrogen removal from low strength anaerobic membrane bioreactor (AnMBR) permeate using complete nitrification and partial denitrification-anammox processes. Frontiers of Environmental Science & Engineering, 18(12): 155

[11] He C S , He P P , Yang H Y , Li L L , Lin Y , Mu Y , Yu H Q . (2017). Impact of zero-valent iron nanoparticles on the activity of anaerobic granular sludge: from macroscopic to microcosmic investigation. Water Research, 127: 32–40

[12] Hoffmann N , Fincheira P , Tortella G , Rubilar O . (2022). The role of iron nanoparticles on anaerobic digestion: mechanisms, limitations, and perspectives. Environmental Science and Pollution Research, 29(55): 82619–82631

[13] Huang X N , Miao X Y , Chu X D , Luo L N , Zhang H Q , Sun Y . (2023). Enhancement effect of biochar addition on anaerobic co-digestion of pig manure and corn straw under biogas slurry circulation. Bioresource Technology, 372: 128654

[14] Jadhav P , Khalid Z B , Zularisam A W , Krishnan S , Nasrullah M . (2022). The role of iron-based nanoparticles (Fe-NPs) on methanogenesis in anaerobic digestion (AD) performance. Environ Res, 112043

[15] Jiang L K , Dang Q L , Zhao X Y , Zhang C Y , Tan X K , Yan Q H . (2022). Mechanism of microbial involvement in nitrogen conversion affecting methane production in dry anaerobic digestion. Journal of Cleaner Production, 369: 133324

[16] Li N , Quan X , Zhuo M , Zhang X , Quan Y , Liang P . (2022). Enhancing methanogenesis of anaerobic granular sludge by incorporating Fe/Fe oxides nanoparticles aided with biofilm disassembly agents and mediating redox activity of extracellular polymer substances. Water Research, 118293

[17] Liang L F , Jin Z , Tao Y , Li Y , Zhao Z Q , Zhang Y B . (2024). Enhanced extracellular electron transfer in magnetite-mediated anaerobic oxidation of methane coupled to humic substances reduction: the pivotal role of membrane-bound electron transfer proteins. Environmental Science & Technology, 58(40): 17756–17765

[18] Lim J W , Chiam J A , Wang J Y . (2014). Microbial community structure reveals how microaeration improves fermentation during anaerobic co-digestion of brown water and food waste. Bioresource Technology, 171: 132–138

[19] Liu H Y , Xu Y , Li L , Li X Y , Dai X H . (2024). Enhancing proton-coupled electron transfer drives efficient methanogenesis in anaerobic digestion. Water Research, 266: 122331

[20] Liu H Y , Xu Y , Li L , Yuan S J , Geng H , Tang Y F , Dai X H . (2022). A novel green composite conductive material enhancing anaerobic digestion of waste activated sludge via improving electron transfer and metabolic activity. Water Research, 220: 118687

[21] Liu L , Chen Y , Qi J , Sun J L , Zhang L G . (2025). The role of sulfidated zero-valent iron in enhancing anaerobic digestion of waste activated sludge. Journal of Environmental Management, 375: 124283

[22] Liu Y , Xiao Q B , Ye X M , Wang C C , Jia Z Y , Du J , Kong X P , Xi Y L . (2021a). Effect of different charged Fe₃O₄ nanoparticles on methane production for anaerobic digestion of wheat straw. Journal of Cleaner Production, 328: 129655

[23] Liu Z H , Zhou A J , Wang S F , Cheng S L , Yin X Y , Yue X P . (2021b). Quorum sensing shaped microbial consortia and enhanced hydrogen recovery from waste activated sludge electro-fermentation on basis of free nitrous acid treatment. Science of the Total Environment, 766: 144348

[24] Lu Y J , Zhou X Q , Zheng Y , Yang H L , Cao W B . (2025). How far do we still need to go with antibiotics in aquatic environments? Antibiotic occurrence, chemical-free or chemical-limited strategies, key challenges, and future perspectives. Water Research, 275: 123179

[25] Lyu Z , Shao N N , Akinyemi T , Whitman W B . (2018). Methanogenesis. Current Biology, 28(13): R727–R732

[26] Mortezaei Y , Williams M R , Demirer G N . (2024). The fate of antibiotic resistance genes during anaerobic digestion of sewage sludge with ultrasonic pretreatment. Environmental Science and Pollution Research, 31(4): 5513–5525

[27] Pan M , Jiang T , Wang Z H , Huang X Z , Liu S J , Huang X M . (2025). Enhanced nitrogen removal via simultaneous nitrification-denitrification (SND) and Feammox in a magnetic zeolite modified intermittently aerated sequential biological reactor. Frontiers of Environmental Science & Engineering, 19(9): 115

[28] Peng H , Zhang Y B , Tan D M , Zhao Z Q , Zhao H M , Quan X . (2018). Roles of magnetite and granular activated carbon in improvement of anaerobic sludge digestion. Bioresource Technology, 249: 666–672

[29] Rani J , Pandey K P , Kushwaha J , Priyadarsini M , Dhoble A S . (2022). Antibiotics in anaerobic digestion: investigative studies on digester performance and microbial diversity. Bioresource Technology, 361: 127662

[30] Shi L , Dong H L , Reguera G , Beyenal H , Lu A H , Liu J , Yu H Q , Fredrickson J K . (2016). Extracellular electron transfer mechanisms between microorganisms and minerals. Nature Reviews Microbiology, 14(10): 651–662

[31] Tang Z , Huang C , Tian Y , Xi B , Guo W , Tan W . (2021). Fate of antibiotic resistance genes in industrial-scale rapid composting of pharmaceutical fermentation residue: the role implications of microbial community structure and mobile genetic elements. Environmental Pollution, 291: 118155

[32] Tao N , Xu M C , Wu X , Pi Z L , Yu C H , Fang D , Zhou L X . (2021). Supplementation of Schwertmannite improves methane produc-tion and heavy metal stabilization during anaerobic swine manure treatment. Fuel, 299: 120883

[33] Wang B Q , Xu Z X , Dong B . (2024a). Occurrence, fate, and ecological risk of antibiotics in wastewater treatment plants in China: a review. Journal of Hazardous Materials, 469: 133925

[34] Wang D B , Pan Q Y , Yang J N , Gong S , Liu X R , Fu Y K . (2024b). Effects of mixtures of engineered nanoparticles and coconta-minants on anaerobic digestion. Environmental Science & Technology, 58: 2598–2614

[35] Wang F Z , Song G , Zhang M , Zhao S N , Wang T , Zhao K , Wang X , Liu R P . (2025). Fe₃O₄ nanoparticles promote methanogenesis in propionate acclimated system. Bioresource Technology, 431: 132608

[36] Wang H P , Sun H , Ren H Y , Cao G L , Xie G J , Xing D F , Ren N Q , Liu B F . (2023a). Metagenomic reveals the methanogenesis metabolic mechanism of high-solids anaerobic digestion of human feces under gradient domestication. Chemical Engineering Journal, 460: 141752

[37] Wang J , Lou Y , Ma D M , Feng K , Chen C , Zhao L , Xing D F . (2023b). Co-treatment with free nitrous acid and calcium peroxide regulates microbiome and metabolic functions of acidogenesis and methanogenesis in sludge anaerobic digestion. Science of the Total Environment, 870: 161924

[38] Wang M W , Ren T F , Yin M X , Lu K C , Xu H , Huang X , Zhang X Y . (2023c). Enhanced anaerobic wastewater treatment by a binary electroactive material: pseudocapacitance/conductance-mediated microbial interspecies electron transfer. Environmental Science & Technology, 57(32): 12072–12082

[39] Xiao K K , Yu Z C , Pei K Y , Sun M , Zhu Y W , Liang S , Hou H J , Liu B C , Hu J P , Yang J K . (2022). Anaerobic digestion of sludge by different pretreatments: changes of amino acids and microbial community. Frontiers of Environmental Science & Engineering, 16(2): 23

[40] Xu H , Wang M W , Hei S , Qi X , Zhang X Y , Liang P , Fu W Y , Pan B C , Huang X . (2024a). Neglected role of iron redox cycle in direct interspecies electron transfer in anaerobic methanogenesis: inspired from biogeochemical processes. Water Research, 262: 122125

[41] Xu J J , Chen X T , Tang R , Feng J W , Yuan S J , Wang W , Hu Z H . (2024b). Removal of pathogenic indicator microorganisms during partial nitrification: the role of free nitrous acid. Frontiers of Environmental Science & Engineering, 18(3): 33

[42] Yamada T , Sekiguchi Y , Imachi H , Kamagata Y , Ohashi A , Harada H . (2005). Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules. Applied and Environmental Microbiology, 71(11): 7493–7503

[43] Yan B B , Chen X , Wang Z , Li J , Li T W , Mu L , Chen G Y . (2025). Anaerobic co-digestion of antibiotic fermentation residues and corn stalks for biogas production and elimination of antibiotic resistance genes. Fuel, 387: 134442

[44] Yang G , Wang J L . (2019). Changes in microbial community structure during dark fermentative hydrogen production. International Journal of Hydrogen Energy, 44(47): 25542–25550

[45] Ye J , Hu A D , Cheng X Y , Lin W F , Liu X , Zhou S G , He Z . (2018a). Response of enhanced sludge methanogenesis by red mud to temperature: spectroscopic and electrochemical elucidation of endogenous redox mediators. Water Research, 143: 240–249

[46] Ye J , Hu A D , Ren G P , Chen M , Tang J H , Zhang P Y , Zhou S G , He Z . (2018b). Enhancing sludge methanogenesis with improved redox activity of extracellular polymeric substances by hematite in red mud. Water Research, 134: 54–62

[47] Yin Y N , Lou T R , Song W Z , Wang C , Wang J L . (2023). Production of medium chain fatty acids from fermentation of antibiotic residuals: fate of antibiotic resistance genes. Bioresource Technology, 379: 129056

[48] Ying G G , He L Y , Ying A J , Zhang Q Q , Liu Y S , Zhao J L . (2017). China must reduce its antibiotic use. Environmental Science & Technology, 51(3): 1072–1073

[49] Zhang J Q , You Z X , Liu D Y , Tang R , Zhao C , Cao Y X , Li F , Song H . (2023a). Conductive proteins-based extracellular electron transfer of electroactive microorganisms. Quantitative Biology, 11(4): 405–420

[50] Zhang X X , Jiao P B , Zhang M , Wu P , Zhang Y F , Wang Y W , Xu K Y , Yu J Z , Ma L P . (2023b). Impacts of organic loading rate and hydraulic retention time on organics degradation, interspecies interactions and functional traits in thermophilic anaerobic co-digestion of food waste and sewage sludge. Bioresource Technology, 370: 128578

[51] Zhang Z Y , Zhang Z H , Zhang C Y , Chang Q , Fang Q X , Liao C M , Chen J B , Alvarez P J J , Chen W , Zhang T . (2024). Simultaneous reduction and methylation of nanoparticulate mercury: the critical role of extracellular electron transfer. Environmental Science & Technology, 58(41): 18368–18378

[52] Zhang Z , Guo L , Wang Y , Zhao Y , She Z , Gao M , Guo Y . (2020). Application of iron oxide (Fe₃O₄) nanoparticles during the two-stage anaerobic digestion with waste sludge: Impact on the biogas production and the substrate metabolism. Renewable Energy, 2724–2735

[53] Zhao Q , Guo W Q , Luo H C , Xing C M , Wang H Z , Liu B H , Si Q S , Ren N Q . (2021). Deciphering the transfers of antibiotic resistance genes under antibiotic exposure conditions: driven by functional modules and bacterial community. Water Research, 205: 117672

[54] Zhao Z Q , Wang J F , Li Y , Zhu T T , Yu Q L , Wang T T , Liang S , Zhang Y B . (2020). Why do DIETers like drinking: metagenomic analysis for methane and energy metabolism during anaerobic digestion with ethanol. Water Research, 171: 115425

[55] Zhao Z Q , Zhang Y B , Yu Q L , Dang Y , Li Y , Quan X . (2016). Communities stimulated with ethanol to perform direct interspecies electron transfer for syntrophic metabolism of propionate and butyrate. Water Research, 102: 475–484

[56] Zhao Z S , Zhang Y B , Li Y , Quan X , Zhao Z Q . (2018). Comparing the mechanisms of ZVI and Fe₃O₄ for promoting waste-activated sludge digestion. Water Research, 144: 126–133

[57] Zhen G Y , Zheng S J , Han Y L , Zhang Z Y , Lu X Q , Xu K Q . (2022). Semi-continuous anolyte circulation to strengthen CO₂ bioelectromethanosynthesis with complex organic matters as the e^–/H⁺ donor for simultaneous biowaste refinery. Chemical Engineering Journal, 430: 133123

[58] Zheng M Q , Ou H , Dong F , He C H , Hu Z H , Wang W . (2023). Mechanism insights into enhanced treatment of wasted activated sludge by hydrogen-mediated anaerobic digestion. Environmental Science and Pollution Research, 30(16): 47787–47799

[59] Zhong Y J , He J G , Zhang P F , Zou X , Pan X L , Zhang J . (2022). Effects of different particle size of zero-valent iron (ZVI) during anaerobic digestion: performance and mechanism from genetic level. Chemical Engineering Journal, 435: 134977

[60] Zhu L R , Wu B C , Liu Y L , Zhang J R , Deng R , Gu L . (2022). Strategy to enhance semi-continuous anaerobic digestion of food waste by combined use of calcium peroxide and magnetite. Water Research, 221: 118801

[61] Zhu R L , Chen Y D , Zhao T , Jiang Q , Wang H Q , Zheng L S , Shi D Z , Zhai J , He Q , Gu L . (2020). Enhanced mesophilic anaerobic co-digestion of waste sludge and food waste by using hematite (α-Fe₂O₃) supported bentonite as additive. Bioresource Technology, 313: 123603

[62] Ziganshina E E , Belostotskiy D E , Bulynina S S , Ziganshin A M . (2021). Effect of magnetite on anaerobic digestion of distillers grains and beet pulp: operation of reactors and microbial community dynamics. Journal of Bioscience and Bioengineering, 131(3): 290–298

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