Periodic Reporting for period 4 - ACETOGENS (Acetogenic bacteria: from basic physiology via gene regulation to application in industrial biotechnology)
Période du rapport: 2022-04-01 au 2023-09-30
Demand for biofuels and other biologically derived chemicals is growing worldwide as efforts increase to reduce dependency on fossil fuels and to limit climate change. Thus, there is an urgent need for sustainable, biotechnological processes to cope with the increasing demand by society for food, water and energy. An innovative solution is to use CO2 or other C1 compounds as feedstock and C1-utilising acetogenic bacteria as the process organisms. Acetogenic bacteria efficiently convert C1 compounds such as CO2, CO, formate or methanol to acetate, some acetogens can also produce ethanol. This makes acetogens ideal biocatalysts for a CO2-based bioeconomy. Gas fermentation using acetogenic bacteria and syngas as feedstock has already been demonstrated in two precommercial 100,000 gal/yr. demonstration facilities deployed at industrial sites to produce fuel ethanol from abundant waste gas resources (by LanzaTech); commercial gas fermentation units are currently in design.
In the project "Acetogens" we aim to better understand the metabolism and its regulation in acetogenic bacteria with a focus on Acetobacterium woodii. In particular we have focussed on formate and methanol metabolism and its regulation, on mixotrophy to enhance carbon recovery, on the role of bacterial microcompartments in substrate conversion and the physiology, regulation and application of carbon capture and hydrogen storage by A. woodii as well as a thermophilic species, Thermoanaerobacter kivui. We have also extended our studies to gut acetogens of the genus Blautia to explore their role in health and well-being of humans and in biotechnology.
Our experiments have given a detailed picture of the biochemistry of methanol conversion in different acetogens and on formate metabolism. By metabolic engineering, we could create a superb acetogenic formate-to-acetate production platform. Moreover, we also redirected carbon flow away from acetate to lactate, formate or butyrate by metabolic engineering and we were able to transform A. woodii into an efficient CO oxidizing bacterium. These studies paved the road for the use of formate, methanol and carbon monoxide as acetogenic feedstocks to produce biocommodities. In addition, a main portion of our project was devoted to the development of a biological process for hydrogen production and scavenging using acetogens. We successfully established procedures to use A. woodii and T. kivui as biorefineries to produce hydrogen from formate and to produce formate from hydrogen plus CO2 with unprecedented rates. These studies culminated in the development of a biobattery or hydrogen storage.
In the project "Acetogens" we aim to better understand the metabolism and its regulation in acetogenic bacteria with a focus on Acetobacterium woodii. In particular we have focussed on formate and methanol metabolism and its regulation, on mixotrophy to enhance carbon recovery, on the role of bacterial microcompartments in substrate conversion and the physiology, regulation and application of carbon capture and hydrogen storage by A. woodii as well as a thermophilic species, Thermoanaerobacter kivui. We have also extended our studies to gut acetogens of the genus Blautia to explore their role in health and well-being of humans and in biotechnology.
Our experiments have given a detailed picture of the biochemistry of methanol conversion in different acetogens and on formate metabolism. By metabolic engineering, we could create a superb acetogenic formate-to-acetate production platform. Moreover, we also redirected carbon flow away from acetate to lactate, formate or butyrate by metabolic engineering and we were able to transform A. woodii into an efficient CO oxidizing bacterium. These studies paved the road for the use of formate, methanol and carbon monoxide as acetogenic feedstocks to produce biocommodities. In addition, a main portion of our project was devoted to the development of a biological process for hydrogen production and scavenging using acetogens. We successfully established procedures to use A. woodii and T. kivui as biorefineries to produce hydrogen from formate and to produce formate from hydrogen plus CO2 with unprecedented rates. These studies culminated in the development of a biobattery or hydrogen storage.
The biochemistry and bioenergetics of acetogenesis from methanol and formate has been deliniated. This is a prerequisite for designing production strains. Expression of the genes encoding bacterial microcompartments has been unravelled and BMCs were enriched and shown to contain products of the 18 gene cluster.
Hydrogen is a promising and widely considered alternative for biofuels and a hydrogen-based economy is a prime solution for a sustainable future. A striking problem of using hydrogen as energy source is the storage and transport due to the physical properties of this highly reactive gas. A promising solution is the hydrogenation of CO2 that leads to formic acid. The hydrogen-dependent CO2 reductase found previously by us in a mesophilic and in a thermophilic acetogen catalyses this reaction and vice versa with highest rates available to date. On the road towards an industrial application we developed a whole-cell-system for both species that efficiently binds H2 to CO2, and large quantities of H2 could be stored in formic by continuous product removal. This reaction is reversible, a prerequisite for a catalyst that produces a storage compound, but also interesting for the production of hydrogen from formate from other sources.
We designed four stirred tank reactor (STR) systems for anaerobic cultivation of microorganisms with a maximum working volume of two liters. One of the recent highlights of our project was the development of a biobattery for hydrogen storage in which binding and release took place in one fermenter. In a paper published in the high impact journal Joule, we demonstrate for the first time that a bio-based system, using Acetobacterium woodii as biocatalyst, allows multiple cycles of bi-directional hydrogenation of CO2 to formic acid in one bioreactor. The process was kept running over two weeks. Unwanted side product formation of acetic acid was prevented through metabolic engineering of the organism. The demonstrated process design can be considered as future “bio-battery” for the reversible storage of electrons in form of H2 in the versatile compound formic acid. This manuscript was very well received by the community but also by public outlets in newspapers, journals and radio broadcasts.
Another highlight was the determined structure of the HDCR. Previously, we had found that the enzyme forms long filaments. Together with the group of Dr. Jan Schuller from the University of Marburg in Germany we solved the structure of the HDCR at atomic resolution by cryo-electron microscopy; these studies revealed the structure of a short HDCR filament from the acetogenic bacterium Thermoanaerobacter kivui at 3.4 Å-resolution. The minimum repeating unit was a hexamer consisting of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize via their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we demonstrate that filamentation and rapid electron transfer through the filament enhances HDCR activity. To investigate the HDCR structure in situ, we imaged T. kivui cells with cryo-electron tomography (together with Prof. ben Engels, University of Bael, Switzerland) and found that HDCR filaments bundle into large ring-shaped superstructures attached to the plasma membrane. This supramolecular organization may further enhance HDCR stability and connectivity to form a specialized metabolic subcompartment within the cell. This study was published in Nature and received much attention from scientists as well as science writers. Further studies on the HDCR will concentrate on the role of the unique cellular compartmentalization of the enzyme.
Hydrogen is a promising and widely considered alternative for biofuels and a hydrogen-based economy is a prime solution for a sustainable future. A striking problem of using hydrogen as energy source is the storage and transport due to the physical properties of this highly reactive gas. A promising solution is the hydrogenation of CO2 that leads to formic acid. The hydrogen-dependent CO2 reductase found previously by us in a mesophilic and in a thermophilic acetogen catalyses this reaction and vice versa with highest rates available to date. On the road towards an industrial application we developed a whole-cell-system for both species that efficiently binds H2 to CO2, and large quantities of H2 could be stored in formic by continuous product removal. This reaction is reversible, a prerequisite for a catalyst that produces a storage compound, but also interesting for the production of hydrogen from formate from other sources.
We designed four stirred tank reactor (STR) systems for anaerobic cultivation of microorganisms with a maximum working volume of two liters. One of the recent highlights of our project was the development of a biobattery for hydrogen storage in which binding and release took place in one fermenter. In a paper published in the high impact journal Joule, we demonstrate for the first time that a bio-based system, using Acetobacterium woodii as biocatalyst, allows multiple cycles of bi-directional hydrogenation of CO2 to formic acid in one bioreactor. The process was kept running over two weeks. Unwanted side product formation of acetic acid was prevented through metabolic engineering of the organism. The demonstrated process design can be considered as future “bio-battery” for the reversible storage of electrons in form of H2 in the versatile compound formic acid. This manuscript was very well received by the community but also by public outlets in newspapers, journals and radio broadcasts.
Another highlight was the determined structure of the HDCR. Previously, we had found that the enzyme forms long filaments. Together with the group of Dr. Jan Schuller from the University of Marburg in Germany we solved the structure of the HDCR at atomic resolution by cryo-electron microscopy; these studies revealed the structure of a short HDCR filament from the acetogenic bacterium Thermoanaerobacter kivui at 3.4 Å-resolution. The minimum repeating unit was a hexamer consisting of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize via their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we demonstrate that filamentation and rapid electron transfer through the filament enhances HDCR activity. To investigate the HDCR structure in situ, we imaged T. kivui cells with cryo-electron tomography (together with Prof. ben Engels, University of Bael, Switzerland) and found that HDCR filaments bundle into large ring-shaped superstructures attached to the plasma membrane. This supramolecular organization may further enhance HDCR stability and connectivity to form a specialized metabolic subcompartment within the cell. This study was published in Nature and received much attention from scientists as well as science writers. Further studies on the HDCR will concentrate on the role of the unique cellular compartmentalization of the enzyme.
All of our data are beyond the state of the art.