Metabolic Engineering Modification and Electricity Production Condition Optimization of Salt-Tolerant Electricigen Shewanella Algae

Abstract

Electricigens are a type of microorganism with extracellular electron transfer ability, which can convert stored chemical energy in organic matter into electrical energy through their own metabolism. Previous studies have shown that electricigens can be used as catalysts to construct microbial fuel cells(MFCs). However, low electricity production remains the most urgent issue for electricigens. This study is based on a salt-tolerant electricigen, Shewanella algae, and uses CRISPRa technology to regulate the expression of pflb and mdh genes in the extracellular electron metabolism pathway, thereby enhancing the ability to produce electricity. Simultaneously, we aim to optimize power generation conditions of Shewanella algae by adding surfactants or electron mediators in the bipolar chamber of fuel cells to enhance electron transfer. The results show that overexpression of pflb and mdh genes increases the current density by 3. 51% and 14. 17%, respectively. The use of surfactants such as Tween-80 increases the current density by approximately 20%-40%. These results indicate that an increase in extracellular electron transfer or electron metabolism can increase power generation, providing a basis for further optimization and transformation of salttolerant electricigen Shewanella algae.

Keywords

Shewanella algae / metabolic engineering / microbial fuel cell(MFC) / surfactant / electron mediator

References

[1] Logan B E, Hamelers B, Rozendal R, et al. Microbial fuel cells: methodology and technology[J]. Environmental Science & Technology, 2006, 40(17): 5181-5192.

[2] Shimoyama T, Komukai S, Yamazawa A, et al. Electricity generation from model organic wastewater in a cassette-electrode microbial fuel cell[J]. Applied Microbiology and Biotechnology, 2008, 80(2): 325-330.

[3] Saran C, Purchase D, Saratale G D, et al. Microbial fuel cell: a green eco-friendly agent for tannery wastewater treatment and simultaneous bioelectricity/power generation[J]. Chemosphere, 2023, 312: 137072.

[4] Wu D G, Zhang B C, Shi S C, et al. Engineering extracellular electron transfer to promote simultaneous brewing wastewater treatment and chromium reduction[J]. Journal of Hazardous Materials, 2024, 465: 133171.

[5] Logan B E. Exoelectrogenic bacteria that powermicrobial fuel cells[J]. Nature ReviewsMicrobiology, 2009, 7(5): 375-381.

[6] Lovley D R. The microbe electric: conversion oforganic matter to electricity[J]. CurrentOpinion in Biotechnology, 2008, 19(6): 564-571.

[7] Myers C R, Myers J M. Localization ofcytochromes to the outer membrane ofanaerobically grown Shewanella putrefaciensMR-1[J]. Journal of Bacteriology, 1992, 174(11): 3429-3438.

[8] Lanthier M, Gregory K B, Lovley D R. Growthwith high planktonic biomass in Shewanellaoneidensis fuel cells[J]. FEMS MicrobiologyLetters, 2008, 278(1): 29-35.

[9] Szeinbaum N, Lin H, Brandes J A, et al. Microbial manganese(Ⅲ)reduction fuelled byanaerobic acetate oxidation[J]. EnvironmentalMicrobiology, 2017, 19(9): 3475-3486.

[10] Ikeda S, Takamatsu Y, Tsuchiya M, et al. Shewanella oneidensis MR-1 as a bacterialplatform for electro-biotechnology[J]. Essays inBiochemistry, 2021, 65(2): 355-364.

[11] Lemaire O N, MÉjean V, Iobbi-Nivol C. TheShewanella genus: ubiquitous organismssustaining and preserving aquatic ecosystems[J]. FEMS Microbiology Reviews, 2020, 44(2):155-170.

[12] Huang Z Z, Yu K Y, Fu S Z, et al. Genomicanalysis reveals high intra-species diversity ofShewanella algae[J]. Microbial Genomics, 2022, 8(2): 000786.

[13] Shi L, Dong H L, Reguera G, et al. Extracellular electron transfer mechanismsbetween microorganisms and minerals[J]. Nature Reviews Microbiology, 2016, 14(10):651-662.

[14] Koeck P J B. Ins and outs of digital electronmicroscopy[J]. Microscopy Research andTechnique, 2000, 49(3): 217-223.

[15] Jones L M, Hanrahan N, Salta M, et al. Towardsimulating offshore oilfield conditions: insightsinto microbiologically influenced corrosion from adual anaerobic biofilm reactor[J]. Applied andEnvironmental Microbiology, 2025, 91(6):e02221-e02224.

[16] Lai B, Tang X H, Li H R, et al. Powerproduction enhancement with a polyanilinemodified anode in microbial fuel cells[J]. Biosensors and Bioelectronics, 2011, 28(1):373-377.

[17] Du Z W, Li H R, Gu T Y. A state of the artreview on microbial fuel cells: a promisingtechnology for wastewater treatment andbioenergy[J]. Biotechnology Advances, 2007, 25(5): 464-482.

[18] Hou H J, Li L, De Figueiredo P, et al. Aircathodemicrobial fuel cell array: a device foridentifying and characterizing electrochemicallyactive microbes[J]. Biosensors andBioelectronics, 2011, 26(5): 2680-2684.

[19] Schröder U, Nießen J, Scholz F. A generation ofmicrobial fuel cells with current outputs boosted by more than one order of magnitude[J]. Angewandte Chemie International Edition, 2003, 42(25): 2880-2883.

[20] YU Y Y, Chen H L, Yong Y C, et al. Conductive artificial biofilm dramaticallyenhances bioelectricity production in Shewanellainoculatedmicrobial fuel cells[J]. ChemicalCommunications, 2011, 47(48): 12825-12827.

[21] Zhang T, Cui C Z, Chen S L, et al. A novelmediatorless microbial fuel cell based on directbiocatalysis of Escherichia coli[J]. ChemicalCommunications, 2006(21): 2257-2259.

[22] Li F, Li Y X, Sun L M, et al. Modularengineering intracellular NADH regenerationboosts extracellular electron transfer ofShewanella oneidensis MR-1[J]. ACS SyntheticBiology, 2018, 7(3): 885-895.

[23] Berríos-Rivera S J, Bennett G N, San K Y. Theeffect of increasing NADH availability on theredistribution of metabolic fluxes in Escherichiacoli chemostat cultures[J]. MetabolicEngineering, 2002, 4(3): 230-237.

[24] San K Y, Bennett G N, Berríos-Rivera S J, et al. Metabolic engineering through cofactormanipulation and its effects on metabolic fluxredistribution in Escherichia coli[J]. MetabolicEngineering, 2002, 4(2): 182-192.

[25] Berríos-Rivera S J, Bennett G N, San K Y. Metabolic engineering of Escherichia coli:increase of NADH availability by overexpressingan NAD+-dependent formate dehydrogenase[J]. Metabolic Engineering, 2002, 4(3): 217-229.

[26] Montoya-Vallejo C, Gil Posada J O, Quintero-Díaz J C. Enhancement of electricity productionin microbial fuel cells using a biosurfactantproducingco-culture[J]. Molecules, 2023, 28(23): 7833.

Funding

Innovation and Entrepreneurship Training Program for College Students, China(202210255065)

Innovation and Entrepreneurship Training Program for College Students, China(117-03-0178012/017)