Bioconversion of C1-gases by mixotrophic co-cultures fermentation with C. carboxidivorans and C. beijerinkii

Abstract

The development of newfangled bioprocess strategies for the capture of C1-gases (CO and CO₂) and their bioconversion into valuable products is currently one of the main focuses of research in order to achieve a more resilient world. This work analyses the viability of the co-culture C. carboxidivorans and C. beijerinkii to produce bioproducts (bioalcohols and organic acids) in mixotrophic conditions. In this way, the bioconversion of C1 gases (CO and CO₂), in the presence of Fe⁰, using mixotrophic co-culture fermentation by C. carboxidivorans and C. beijerinkii, was evaluated, analyzing the influence of the ratio between both microorganisms, the pH, and the presence of Fe⁰. As a result, up to 7 g/L of butanol were achieved at pH 7, 12.5 g/L Fe⁰, and using a 1:1 ratio of C. carboxidivorans: C. beijerinkii, also improving the production of ethanol, acetic acid, and butyric acid as compared to individual culture fermentations. Finally, the operation in a bioreactor, comparing discontinuous and continuous gas feeding operation modes, was also studied, with better C1-gases utilization and overall fermentation efficiency (7 vs 4.6 g/L butanol) in continuous gas operation mode.

Keywords

C1-gases conversion / Mixotrophic fermentation / Fe⁰ / Co-culture / Clostridia spp.

References

[1] AntonicelliG, RicciL, TarraranL, et al. . Expanding the product portfolio of carbon dioxide and hydrogen-based gas fermentation with an evolved strain of Clostridium carboxidivorans. Bioresour Technol, 2023, 387. 129689

[2] ArslanK, VeigaMC, KennesC. Autotrophic (C1-gas) versus heterotrophic (fructose) accumulation of acetic acid and ethanol in Clostridium aceticum. Bioresour Technol, 2021, 337. 125485

[3] BayarB, VeigaMC, KennesC. Bioproduction of acetic acid from carbon dioxide as single substrate and zero valent iron (ZVI) by clostridia. J CO2 Util, 2022, 58: 101915.

[4] BenevenutiC, BotelhoA, RibeiroR, et al. . Experimental design to improve cell growth and ethanol production in syngas fermentation by Clostridium carboxidivorans. Catalysts, 2020, 10: 59.

[5] BenevenutiC, BrancoM, Do Nascimento-CorreaM, et al. . Residual gas for ethanol production by Clostridium carboxidivorans in a dual impeller stirred tank bioreactor (STBR). Fermentation, 2021, 7: 199.

[6] BranskaB, PechacovaZ, KolekJ, et al. . Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions. Biotechnol Biofuels, 2018, 11: 99.

[7] CaiD, ChenH, ChenC, et al. . Gas stripping–pervaporation hybrid process for energy-saving product recovery from acetone–butanol–ethanol (ABE) fermentation broth. Chem Eng J, 2016, 287: 1-10.

[8] ChengC, ShaoY, LiW, et al. . Electricity-enhanced anaerobic, non-photosynthetic mixotrophy by Clostridium carboxidivorans with increased carbon efficiency and alcohol production. Energy Convers Manag, 2022, 252. 115118

[9] DashS, NgCY, MaranasCD. Metabolic modeling of clostridia: current developments and applications. FEMS Microbiol Lett, 2016, 3634fnw004.

[10] dos VieiraCF, S, Codogno MC, Maugeri Filho F, , et al. . Sugarcane bagasse hydrolysates as feedstock to produce the isopropanol-butanol-ethanol fuel mixture: Effect of lactic acid derived from microbial contamination on Clostridium beijerinckii DSM 6423. Bioresour Technol, 2021, 319. 124140

[11] DuY, ZouW, ZhangK, et al. . Advances and Applications of Clostridium co-culture systems in biotechnology. Front Microbiol, 2020, 11: 1-22.

[12] Fernández-BlancoC, VeigaMC, KennesC. Efficient production of n-caproate from syngas by a co-culture of Clostridium aceticum and Clostridium kluyveri. J Environ Managet, 2022, 302. 113992

[13] Fernández-BlancoC, Robles-IglesiasR, Naveira-PazosC, et al. . Production of biofuels from C1-gases with Clostridium and related bacteria—Recent advances. Microb Biotechnol, 2023, 16: 726-741.

[14] Fernández-NaveiraÁ, AbubackarHN, VeigaMC, et al. . Efficient butanol-ethanol (B-E) production from carbon monoxide fermentation by Clostridium carboxidivorans. Appl Microbiol Biotechnol, 2016, 100: 3361-3370.

[15] Fernández-NaveiraÁ, AbubackarHN, VeigaMC, et al. . Production of chemicals from C1 gases (CO, CO₂) by Clostridium carboxidivorans. World J Microbiol Biotechnol, 2017, 33343.

[16] Fernández-NaveiraÁ, VeigaMC, KennesC. Glucose bioconversion profile in the syngas-metabolizing species Clostridium carboxidivorans. Bioresour Technol, 2017, 244: 552-559.

[17] Fernández-NaveiraÁ, VeigaMC, KennesC. H-B-E (hexanol-butanol-ethanol) fermentation for the production of higher alcohols from syngas/waste gas. J Chem Technol Biotechnol, 2017, 92: 712-731.

[18] Fernández-NaveiraÁ, VeigaMC, KennesC. Selective anaerobic fermentation of syngas into either C2–C6 organic acids or ethanol and higher alcohols. Bioresour Technol, 2019, 280: 387-395.

[19] GuptaR, MishraA, ThirupathaiahY, ChandelAK. Biochemical conversion of CO₂ in fuels and chemicals: status, innovation, and industrial aspects. Biomass Convers Biorefin, 2022, 14: 3007-3030.

[20] HazeenaSH, Nair SaliniC, SindhuR, PandeyA, BinodP. Simultaneous saccharification and fermentation of oil palm front for the production of 2,3-butanediol. Bioresour Technol, 2019, 278: 145-149.

[21] HeY, LensPNL, VeigaMC, KennesC. Selective butanol production from carbon monoxide by an enriched anaerobic culture. Sci Total Environ, 2022, 806. 150579

[22] HwangY, SivagurunathanP, LeeMK, et al. . Enhanced hydrogen fermentation by zero valent iron addition. Int J Hydrogen Energy, 2019, 44: 3387-3394.

[23] JiangW, Hernández VillamorD, PengH, et al. . Metabolic engineering strategies to enable microbial utilization of C1 feedstocks. Nat Chem Biol, 2021, 178845-855.

[24] JonesSW, FastAG, CarlsonED, et al. . CO₂ fixation by anaerobic non-photosynthetic mixotrophy for improved carbon conversion. Nat Commun, 2016, 7: 12800.

[25] LanzilloF, RuggieroG, RaganatiF, et al. . Batch syngas fermentation by Clostridium carboxidivorans for production of acids and alcohols. Processes, 2020, 8: 1075.

[26] LanzilloF, PisacaneS, CapillaM, et al. . Continuous H-B-E fermentation by Clostridium carboxidivorans: CO vs syngas. N Biotechnol, 2024, 81: 1-9.

[27] LiewF, HenstraAM, WinzerK, et al. . Insights into CO₂ fixation pathway of Clostridium autoethanogenum by targeted mutagenesis. Mbio, 2016.

[28] LittyD, MüllerV. Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source. Microb Biotechnol, 2021, 14: 2686-2692.

[29] LiuK, AtiyehHK, StevensonBS, et al. . Mixed culture syngas fermentation and conversion of carboxylic acids into alcohols. Bioresour Technol, 2014, 152: 337-346.

[30] López-LinaresJC, García-CuberoMT, LucasS, CocaM. Integral valorization of cellulosic and hemicellulosic sugars for biobutanol production: ABE fermentation of the whole slurry from microwave pretreated brewer’s spent grain. Biomass Bioenerg, 2020, 135. 105524

[31] López-LinaresJC, García-CuberoMT, CocaM, LucasS. Efficient biobutanol production by acetone-butanol-ethanol fermentation from spent coffee grounds with microwave assisted dilute sulfuric acid pretreatment. Bioresour Technol, 2021, 320. 124348

[32] López-LinaresJC, CocaM, PlazaPE, LucasS, García-CuberoMT. Waste-to-fuel technologies for the bioconversion of carrot discards into biobutanol. Renew Energy, 2023, 202: 362-369.

[33] MolitorB, RichterH, MartinME, et al. . Carbon recovery by fermentation of CO-rich off gases - Turning steel mills into biorefineries. Bioresour Technol, 2016, 215: 386-396.

[34] NOAA. DIALOG. 2023. https://www.noaa.gov/research. Accessed 17 Oct 2024.

[35] PlazaPE, Gallego-MoralesLJ, Peñuela-VásquezM, et al. . Biobutanol production from brewer’s spent grain hydrolysates by Clostridium beijerinckii. Bioresour Technol, 2017, 244: 166-174.

[36] QiG, XiongL, LiH, et al. . Hydrotropic pretreatment on wheat straw for efficient biobutanol production. Biomass Bioenerg, 2019, 122: 76-83.

[37] SalehizadehH, YanN, FarnoodR. Recent advances in microbial CO₂ fixation and conversion to value-added products. Chem Eng J, 2020, 390. 124584

[38] SchuchmannK, MüllerV. Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria. Nat Rev Microbiol, 2014, 12: 809-821.

[39] SchulzS, MolitorB, AngenentLT. Acetate augmentation boosts the ethanol production rate and specificity by Clostridium ljungdahlii during gas fermentation with pure carbon monoxide. Bioresour Technol, 2023, 369: 128387.

[40] SivalingamV, ParhizkarabyanehP, WinklerD, et al. . Impact of electrochemical reducing power on homoacetogenesis. Bioresour Technol, 2022, 345: 126512.

[41] SunX, AtiyehHK, KumarA, et al. . Biochar enhanced ethanol and butanol production by Clostridium carboxidivorans from syngas. Bioresour Technol, 2018, 265: 128-138.

[42] TakemuraK, KatoJ, KatoS, et al. . Autotrophic growth and ethanol production enabled by diverting acetate flux in the metabolically engineered Moorella thermoacetica. J Biosci Bioeng, 2021, 132: 569-574.

[43] ThanapornsinT, SirisantimethakomL, LaopaiboonL, LaopaiboonP. Effectiveness of low-cost bioreactors integrated with a gas stripping system for butanol fermentation from sugarcane molasses by Clostridium beijerinckii. Fermentation, 2022, 8: 214.

[44] ThunuguntlaR, AtiyehHK, HuhnkeRL, TannerRS. CO₂-based production of C2–C6 acids and alcohols: The potential of novel Clostridia. Bioresour Technol Rep, 2024, 25. 101713

[45] ThunuguntlaR, AtiyehHK, ZhangH, et al. . Biochar facilitated biological CO₂ conversion to C2–C6 alcohols and fatty acids. Bioresour Technol, 2024, 397: 130464.

[46] VeesCA, HerwigC, PflüglS. Mixotrophic co-utilization of glucose and carbon monoxide boosts ethanol and butanol productivity of continuous Clostridium carboxidivorans cultures. Bioresour Technol, 2022, 353. 127138

[47] WangP, ChenYM, WangY, et al. . Towards comprehensive lignocellulosic biomass utilization for bioenergy production: Efficient biobutanol production from acetic acid pretreated switchgrass with Clostridium saccharoperbutylacetonicum N1–4. Appl Energy, 2019, 236: 551-559.

[48] WuP, ZhangJ, LiJ, et al. . Deciphering the role and mechanism of nano zero-valent iron on medium chain fatty acids production from CO₂ via chain elongation in microbial electrosynthesis. Sci Total Environ, 2023, 863: 160898.

[49] XueC, ChengC. Butanol Production by Clostridium. Adv Bioenerg, 2019, 4: 35-77.

[50] YangY, NieX, JiangY, et al. . Metabolic regulation in solventogenic clostridia: regulators, mechanisms and engineering. Biotechnol Adv, 2018, 36: 905-914.

[51] YangZ, LeeroDD, YinC, et al. . Clostridium as microbial cell factory to enable the sustainable utilization of three generations of feedstocks. Bioresour Technol, 2022, 361. 127656

[52] ZhangJ, TaylorS, WangY. Effects of end products on fermentation profiles in Clostridium carboxidivorans P7 for syngas fermentation. Bioresour Technol, 2016, 218: 1055-1063.

[53] ZhangS, QuC, HuangX, et al. . Enhanced isopropanol and n -butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate. J Ind Microbiol Biotechnol, 2016, 43: 915-925.

Funding

Ministerio de Ciencia e Innovación(TED2021-129826A-I00/AEI/10.13039/501100011033)

Consejería de Educación, Junta de Castilla y León(UIC 320)

Universidad de Valladolid(UVa postdoctoral)

RIGHTS & PERMISSIONS

The Author(s)