Towards Syngas Electro-fermentation: Utilizing and improving Clostridium ljungdahlii as a host for microbial electrosynthesis
Doctoral thesis, 2025
Gas fermentation technology using acetogenic bacteria couples the capture of greenhouse gases (e.g. CO2 and CO) with production of value-added chemicals from syngas. However, CO2 and CO fixation by acetogens via the Wood-Ljungdahl pathway requires more reducing equivalents than those present in syngas. The intracellular redox balance of electro-active bacteria can be regulated by a bioelectrochemical system (BES), which combines microorganisms such as acetogenic bacteria with an electrochemical system. The electro-fermentation of CO2 and CO in a BES has been successfullydemonstrated, yet its applicability remains unexplored.
Optimizing the operational parameters for the host strain in a BES is essential to ensure the desired performance. At present, low ATP yield from metabolism, pH sensitivity, and inefficient electron uptake in a BES limits cell growth and product formation of acetogens during electro-fermentation. This can be ameliorated only by detailed understanding of their physiology and extracellular electron transfer.
The aims of this thesis were 1) to identify the most important gas-fermenting bacteria for CO electro-fermentation, and 2) to improve their performance via parameter optimization and strain engineering using adaptive laboratory evolution (ALE) and rational design.
Clostridium ljungdahlii, which is a proven electro-active acetogen, was selected for microbial electrosynthesis. Clostridium autoethanogenum, which is genetically similar to C. ljungdahlii, was identified as the dominant species in aCO-enriched culture originating from cow fecal waste and meant for CO electro-fermentation. Low current input (10 mA) in a BES promoted ethanol production and improved cell viability. Operational parameters and culture conditions were systematically tested for improved performance and reproducibility. ALE of C. ljungdahlii on iron was attempted to identify and improve potential mechanisms of extracellular electron uptake. Genome sequencing following ALE revealedmutations in membrane-related proteins. A mutant was found to produce ethanol, hypothetically as an energy storage chemical, rather than enhancing extracellular electron uptake and producing acetate. Heterologous expression of pyruvate formate lyase from Acetobacterium woodii improved growth and acetate production of C. ljungdahlii under H2:CO2conditions both in the absence and presence of formate as a co-substrate.
The results of this thesis will help ameliorate CO2 and CO capture by combined gas fermentation and electro-fermentation.
Optimizing the operational parameters for the host strain in a BES is essential to ensure the desired performance. At present, low ATP yield from metabolism, pH sensitivity, and inefficient electron uptake in a BES limits cell growth and product formation of acetogens during electro-fermentation. This can be ameliorated only by detailed understanding of their physiology and extracellular electron transfer.
The aims of this thesis were 1) to identify the most important gas-fermenting bacteria for CO electro-fermentation, and 2) to improve their performance via parameter optimization and strain engineering using adaptive laboratory evolution (ALE) and rational design.
Clostridium ljungdahlii, which is a proven electro-active acetogen, was selected for microbial electrosynthesis. Clostridium autoethanogenum, which is genetically similar to C. ljungdahlii, was identified as the dominant species in aCO-enriched culture originating from cow fecal waste and meant for CO electro-fermentation. Low current input (10 mA) in a BES promoted ethanol production and improved cell viability. Operational parameters and culture conditions were systematically tested for improved performance and reproducibility. ALE of C. ljungdahlii on iron was attempted to identify and improve potential mechanisms of extracellular electron uptake. Genome sequencing following ALE revealedmutations in membrane-related proteins. A mutant was found to produce ethanol, hypothetically as an energy storage chemical, rather than enhancing extracellular electron uptake and producing acetate. Heterologous expression of pyruvate formate lyase from Acetobacterium woodii improved growth and acetate production of C. ljungdahlii under H2:CO2conditions both in the absence and presence of formate as a co-substrate.
The results of this thesis will help ameliorate CO2 and CO capture by combined gas fermentation and electro-fermentation.
Adaptive laboratory evolution
Clostridium ljungdahlii
Gas fermentation
Pyruvate formate lyase
Microbial electrosynthesis
Electro-fermentation
Metabolic engineering
Hall 10:an, 10th floor Chemistry Building, Kemigården 4, Chalmers University of Technology
Opponent: Prof. Dr. ir. Korneel Rabaey, Department of Biotechnology, Ghent University, Belgium
Author
Chaeho Im
Chalmers, Life Sciences, Industrial Biotechnology
Clostridium ljungdahlii as a biocatalyst in microbial electrosynthesis – Effect of culture conditions on product formation
Bioresource Technology Reports,;Vol. 19(2022)
Journal article
Low electric current in a bioelectrochemical system facilitates ethanol production from CO using CO-enriched mixed culture
FRONTIERS IN MICROBIOLOGY,;Vol. 15(2024)
Journal article
Im, C, Valgepea, K, Modin, O, Nygård, Y, Franzén, C. J. Different growth pattern during microbial electrosynthesis using C. ljungdahlii evolutionary adapted on iron
Im, C, Krige, A, Valgepea, K, Oskar Modin, Nygård, Y, Franzén C. J. Heterologous expression of pyruvate formate lyase in Clostridium ljungdahlii enhances cell growth in a bioelectrochemical system
Ancient Earth was abundant in carbon-based gases, such as CO2 and CO, in the atmosphere, due to volcanic activities, along with metals present on the ground. An ancient form of life, so-called acetogenic bacteria, are bacteria that thrived by utilizing these gaseous carbon compounds long before other nutrients became available for their growth. This ancient metabolic pathway, which uses gaseous carbons, has found modern applications in alternative fuel production, addressing the pressing challenge of global warming caused by extensive fossil fuel use. Another recently discovered ancient form of life, electro-active bacteria, thrived by utilizing insoluble oxidized metals for respiration, akin to how humans rely on oxygen. The discovery of this metabolic process has facilitated its integration with existing electrochemical knowledge, leading to the emergence of bioelectrochemical systems (BESs).
This research focuses on the integration of gas fermentation and a bioelectrochemical system (BES) into gas electro-fermentation to address methods for alternative fuel production and to reduce greenhouse gas emissions. The BES utilizes electro-active bacteria, which interact with electrodes in electrochemical cells connected to an external electric power supply. The study achieved improved efficiency using the acetogen Clostridium ljungdahlii in a BES. CO-enriched cow fecal waste was shown to contain an acetogen similar to C. ljungdahlii, which could be utilized to produce ethanol with a higher yield during CO electro-fermentation compared to fermentation without the application of an electric current. Consequently, the study confirmed that it was appropriate to select the well-known electro-active acetogen C. ljungdahlii as the host organism for CO electro-fermentation.
To optimize the performance of the BES, electrode potentials and culture conditions were optimized. This resulted in reproducible performance and enhanced growth of C. ljungdahlii in the BES. Furthermore, to improve the performance of C. ljungdahlii in a BES, it was cultured on iron, which aimed to improve its interaction with electrodes in a BES. The evolved C. ljungdahlii strain in the BES produced more formate and ethanol, with reduced acetate production and lower cell growth, compared to the wild-type strain. Finally, C. ljungdahlii was genetically engineered to improve cell growth, addressing the issue of low cell density in the BES. The genetically engineered C. ljungdahlii strain demonstrated significantly improved growth during both gas fermentation and microbial electrosynthesis.
In summary, this research shows that syngas electro-fermentation can be a way to produce fuels and chemicals from CO2 which will help to reduce greenhouse gas emissions.
This research focuses on the integration of gas fermentation and a bioelectrochemical system (BES) into gas electro-fermentation to address methods for alternative fuel production and to reduce greenhouse gas emissions. The BES utilizes electro-active bacteria, which interact with electrodes in electrochemical cells connected to an external electric power supply. The study achieved improved efficiency using the acetogen Clostridium ljungdahlii in a BES. CO-enriched cow fecal waste was shown to contain an acetogen similar to C. ljungdahlii, which could be utilized to produce ethanol with a higher yield during CO electro-fermentation compared to fermentation without the application of an electric current. Consequently, the study confirmed that it was appropriate to select the well-known electro-active acetogen C. ljungdahlii as the host organism for CO electro-fermentation.
To optimize the performance of the BES, electrode potentials and culture conditions were optimized. This resulted in reproducible performance and enhanced growth of C. ljungdahlii in the BES. Furthermore, to improve the performance of C. ljungdahlii in a BES, it was cultured on iron, which aimed to improve its interaction with electrodes in a BES. The evolved C. ljungdahlii strain in the BES produced more formate and ethanol, with reduced acetate production and lower cell growth, compared to the wild-type strain. Finally, C. ljungdahlii was genetically engineered to improve cell growth, addressing the issue of low cell density in the BES. The genetically engineered C. ljungdahlii strain demonstrated significantly improved growth during both gas fermentation and microbial electrosynthesis.
In summary, this research shows that syngas electro-fermentation can be a way to produce fuels and chemicals from CO2 which will help to reduce greenhouse gas emissions.
Elucidating stress related to fermenting syngas – towards efficient conversion of CO and CO2 into bioethanol
Swedish Energy Agency (46605-1), 2019-04-01 -- 2023-03-31.
Subject Categories (SSIF 2025)
Industrial Biotechnology
Areas of Advance
Life Science Engineering (2010-2018)
ISBN
978-91-8103-134-8
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5592
Publisher
Chalmers
Hall 10:an, 10th floor Chemistry Building, Kemigården 4, Chalmers University of Technology
Opponent: Prof. Dr. ir. Korneel Rabaey, Department of Biotechnology, Ghent University, Belgium