Continuous sulfide supply enhanced autotrophic production of alcohols with Clostridium ragsdalei

Abstract

Autotrophic syngas fermentation with clostridia enables the conversion of CO, CO₂, and H₂ into organic acids and alcohols. The batch process performance of Clostridium ragsdalei was studied in fully controlled and continuously gassed (600 mbar CO, 200 mbar H₂, 200 mbar CO₂) stirred-tank bioreactors. The final ethanol concentration varied as function of the reaction conditions. Decreasing the pH from pH 6.0–5.5 at a temperature of 37 °C increased the ethanol concentration from 2.33 g L⁻¹ to 3.95 g L⁻¹, whereas lowering the temperature from 37 to 32 °C at constant pH 6.0 resulted in a final ethanol concentration of 5.34 g L⁻¹ after 5 days of batch operation. The sulphur availability was monitored by measuring the cysteine concentration in the medium and the H₂S fraction in the exhaust gas. It was found that most of the initially added sulphur was stripped out within the first day of the batch process (first half of the exponential growth phase). A continuous sodium sulfide feed allowed ethanol concentrations to increase more than threefold to 7.67 g L⁻¹ and the alcohol-to-acetate ratio to increase 43-fold to 17.71 g g⁻¹.

Keywords

Clostridium ragsdalei / Syngas fermentation / Alcohol production / Sulfur limitation

References

[1] Abubackar HN, Veiga MC, Kennes C. Biological conversion of carbon monoxide to ethanol: effect of pH, gas pressure, reducing agent and yeast extract. Bioresour Technol, 2012, 114: 518-522.

[2] Abubackar HN, Fernández-Naveira Á, Veiga MC, Kennes C. Impact of cyclic pH shifts on carbon monoxide fermentation to ethanol by Clostridium autoethanogenum. Fuel, 2016, 178: 56-62.

[3] Aitken A, Learmonth M. Walker JM. Estimation of disulfide bonds using Ellman’s reagent. The protein protocols handbook, 2009, Totowa: Humana Press.

[4] Aklujkar M, Leang C, Shrestha PM, Shrestha M, Lovley DR. Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H₂ and CO₂ compared to organotrophic growth with fructose. Sci Rep, 2017

[5] Bengelsdorf FR, Poehlein A, Linder S, Erz C, Hummel T, Hoffmeister S, Daniel R, Dürre P. Industrial acetogenic biocatalysts: a comparative metabolic and genomic analysis. Front Microbiol, 2016

[6] Bengelsdorf FR, Beck MH, Erz C, Hoffmeister S, Karl MM, Riegler P, Wirth S, Poehlein A, Weuster-Botz D, Dürre P. Bacterial anaerobic synthesis gas (Syngas) and CO₂ + H₂ fermentation. Adv Appl Microbiol, 2018, 103: 143-221.

[7] Devarapalli M, Atiyeh HK, Phillips JR, Lewis RS, Huhnke RL. Ethanol production during semi-continuous syngas fermentation in a trickle bed reactor using Clostridium ragsdalei. Bioresour Technol, 2016, 209: 56-65.

[8] Diender M, Stams AJM, Sousa DZ. Pathways and bioenergetics of anaerobic carbon monoxide fermentation. Front Microbiol, 2015

[9] Doll K, Rückel A, Kämpf P, Wende M, Weuster-Botz D. Two stirred-tank bioreactors in series enable continuous production of alcohols from carbon monoxide with Clostridium carboxidivorans. Bioprocess Biosyst Eng, 2018, 41: 1403-1416.

[10] Griffin DW, Schultz MA. Fuel and chemical products from biomass syngas: a comparison of gas fermentation to thermochemical conversion routes. Environ Prog Sustain Energy, 2012, 31: 219-224.

[11] Groher A, Weuster-Botz D. Comparative reaction engineering analysis of different acetogenic bacteria for gas fermentation. J Biotechnol, 2016, 228: 82-94.

[12] Gu H, Yang Y, Wang M, Chen S, Wang H, Li S, Ma Y, Wang J. Novel cysteine desulfidase CdsB involved in releasing cysteine repression of toxin synthesis in Clostridium difficile. Front Cell Infect Microbiol, 2017

[13] Hermann M, Teleki A, Weitz S, Niess A, Freund A, Bengelsdorf FR, Takors R. Electron availability in CO₂, CO and H₂ mixtures constrains flux distribution, energy management and product formation in Clostridium ljungdahlii. Microb Biotechnol, 2020, 6: 1831-1846.

[14] Hu P, Bowen SH, Lewis RS. A thermodynamic analysis of electron production during syngas fermentation. Bioresour Technol, 2011, 102: 8071-8076.

[15] Huhnke RL, Lewis RS, Tanner RS (2010) Isolation and characterization of novel clostridial species. US Patent 7,704,723. 27 Apr 2010

[16] Hurst KM, Lewis RS. Carbon monoxide partial pressure effects on the metabolic process of syngas fermentation. Biochem Eng J, 2010, 48(2): 159-165.

[17] Infantes A, Kugel M, Neumann A. Evaluation of media components and process parameters in a sensitive and robust fed-batch syngas fermentation system with Clostridium ljungdahlii. Fermentation, 2020

[18] Köpke M, Mihalcea C, Liew F, Tizard JH, Ali MS, Conolly JJ, Al-Sinawi B, Simpson SD. 2,3-butanediol production by acetogenic bacteria, an alternative route to chemical synthesis, using industrial waste gas. Appl Environ Microbiol, 2011, 77: 5467-5475.

[19] Köpke M, Gerth ML, Maddock DJ, Mueller AP, Liew F, Simpson SD, Patrick WM. Reconstruction of an acetogenic 2,3-butanediol pathway involving a novel NADPH-dependent primary-secondary alcohol dehydrogenase. Appl Environ Microbiol, 2014, 80: 3394-3403.

[20] Kundiyana DK, Huhnke RL, Wilkins MR. Syngas fermentation in a 100-L pilot scale fermentor: design and process considerations. J Biosci Bioeng, 2010, 109: 492-498.

[21] Kundiyana DK, Wilkins MR, Maddipati P, Huhnke RL. Effect of temperature, pH and buffer presence on ethanol production from synthesis gas by “Clostridium ragsdalei”. Bioresour Technol, 2011, 102: 5794-5799.

[22] Lee J, Lee JW, Chae CG, Kwon SJ, Kim YJ, Lee JH, Lee HS. Domestication of the novel alcohologenic acetogen Clostridium sp. AWRP: from isolation to characterization for syngas fermentation. Biotechnol Biofuels, 2019

[23] Liakakou ET, Infantes A, Neumann A, Vreugdenhil BJ. Connecting gasification with syngas fermentation: comparison of the performance of lignin and beech wood. Fuel, 2021

[24] Maddipati P, Atiyeh HK, Bellmer DD, Huhnke RL. Ethanol production from syngas by Clostridium strain P11 using corn steep liquor as a nutrient replacement to yeast extract. Bioresour Technol, 2011, 102: 6494-6501.

[25] Maddox IS, Steiner E, Hirsch S, Wessner S, Gutierrez NA, Gapes JR, Schuster KC. The cause of “acid-crash” and “acidogenic fermentations” during the batch acetone-butanol-ethanol (ABE-) fermentation process. J Mol Microbiol Biotechnol, 2000, 2: 95-100.

[26] Ntagia E, Chatzigiannidou I, Williamson AJ, Arends JBA, Rabaey K. Homoacetogenesis and microbial community composition are shaped by pH and total sulfide concentration. Microb Biotechnol, 2020, 13(4): 1026-1038.

[27] Ragsdale SW. Enzymology of the Wood–Ljungdahl pathway of acetogenesis. Ann NY Acad Sci, 2008, 1125: 129-136.

[28] Ramió-Pujol S, Ganigué R, Bañeras L, Colprim J. How can alcohol production be improved in carboxydotrophic clostridia?. Process Biochem, 2015, 50: 1047-1055.

[29] Ramió-Pujol S, Ganigué R, Bañeras L, Colprim J. Incubation at 25 °C prevents acid crash and enhances alcohol production in Clostridium carboxidivorans P7. Bioresour Technol, 2015, 192: 296-303.

[30] Richter H, Martin M, Angenent L. A two-stage continuous fermentation system for conversion of syngas into ethanol. Energies, 2013, 6: 3987-4000.

[31] Richter H, Molitor B, Diender M, Sousa DZ, Angenent LT. A narrow pH range supports butanol, hexanol, and octanol production from syngas in a continuous co-culture of Clostridium ljungdahlii and Clostridium kluyveri with in-line product extraction. Front Microbiol, 2016

[32] Richter H, Molitor B, Wei H, Chen W, Aristilde L, Angenent LT. Ethanol production in syngas-fermenting Clostridium ljungdahlii is controlled by thermodynamics rather than by enzyme expression. Energy Environ Sci, 2016, 9: 2392-2399.

[33] Rückel A, Hannemann J, Maierhofer C, Fuchs A, Weuster-Botz D. Studies on syngas fermentation with Clostridium carboxidivorans in stirred-tank reactors with defined gas impurities. Front Microbiol, 2021

[34] Schuchmann K, Müller V. Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria. Nat Rev Microbiol, 2014, 12: 809-821.

[35] Shen S, Wang G, Zhang M, Tang Y, Gu Y, Jiang W, Wang Y, Zhuang Y. Effect of temperature and surfactant on biomass growth and higher-alcohol production during syngas fermentation by Clostridium carboxidivorans P7. Bioresour Bioprocess, 2020, 7: 56.

[36] Valgepea K, Lemgruber RSP, Meaghan K, Palfreyman RW, Abdalla T, Heijstra BD, Behrendorff JB, Tappel R, Köpke M, Simpson SD, Nielsen LK, Marcellin E. Maintenance of ATP homeostasis triggers metabolic shifts in gas-fermenting acetogens. Cell Syst, 2017, 4: 505-515.

[37] Wang S, Zhang Y, Dong H, Mao S, Zhu Y, Wang R, Luan G, Li Y. Formic acid triggers the “Acid Crash” of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum. Appl Environ Microbiol, 2011, 77: 1674-1680.

[38] Zhu HF, Liu ZY, Zhou X, Yi JH, Lun ZM, Wang SN, Tang WZ, Li FL. Energy conservation and carbon flux distribution during fermentation of CO or H₂/CO₂ by Clostridium ljungdahlii. Front Microbiol, 2020

Funding

Bundesministerium für Bildung und Forschung(031B0677A)