Development of a Scalable Reactor for Bioelectromethanogenesis and other Bioelectrochemical Applications zur Erlangung des akademischen Grades einer DOKTORIN DER INGENIEURWISSENSCHAFTEN von der KIT-Fakultät für Chemieingenieurwesen und Verfahrenstechnik des Karlsruher Instituts für Technologie (KIT) genehmigte DISSERTATION von Franziska Enzmann Tag der mündlichen Prüfung: 17.12.2019 Erstgutachter/-in: Prof. Dr. Christoph Syldatk Zweitgutachter/-in: Prof. Dr. Clemens Posten Acknowledgements I Acknowledgements This doctoral thesis was developed between 2016 and 2019 at the DECHEMA Research Institute as part of the project MIKE, funded by the Bundesministerium für Bildung und Forschung (BMBF). The work was supervised by Prof. Dr. Christoph Syldatk (Karlsuher Institute of Technology) and Dr. Dirk Holtmann (DECHEMA Research Institute). I wish to thank both of them for their constructive support over the course of the project. Important support was also rendered by Dr. Florian Mayer and all members of the industrial biotechnology research group, including Denise Gronemeier and Marco Genuardi, who prepared their Bachelor thesis based on the project. Cora Kroner and Dr. Markus Stöckl helped a lot with electrochemical issues and Dr. Julia Patzsch with the physisorptions measurements. Andreas Butterhof organized the pipe for the pilot plant reactor. I wish to thank all of them for their collaboration. Special thanks to the DECHEMA workshop team, especially Henry Kopietz, Yvonne Hohmann and Jano Bender, for the construction of the reactors. Thanks to the Studienstiftung des Deutschen Volkes, especially Prof. Reinhard Fischer and the Karlsruher house of youg scientists (KYHS) for their support during my complete studies and especially during this project. Thanks to my family, especially my partner Jochen, for the continuous support. Publications and Presentations II Publications and Presentations Original Research Papers F. Enzmann, F. Mayer, M. Stöckl, K.-M. Mangold, R. Hommel, D. Holtmann, „Transferring bioelectrochemical processes from H-cells to a scalable bubble column reactor,” Chemical Engineering Science, vol. 193, pp. 133-143, 2019. T. Krieg, F. Enzmann, D. Sell, J. Schrader, D. Holtmann, “Simulation of the current generation of a microbial fuel cell in a laboratory wastewater treatment plant,” Appl. Energy, vol. 195, pp. 942–949, Jun. 2017. Mayer, F., Enzmann, F., Lopez, A.M., Holtmann, D., “Performance of different methanogenic species for the microbial electrosynthesis of methane from carbon dioxide,” Bioresour. Technol. 121706, 2019. F. Enzmann, F. Mayer, D. Holtmann, „Process parameters influence the extracellular electron transfer mechanism in bioelectromethanogenesis,” Int. J. Hydrogen Energy, accepted for publication 4.07.2019. Review Articles and Book Chapters F. Enzmann, M. Stöckl, A.-P. Zeng, D. Holtmann, “Same but different - Scale-Up and numbering up in electrobiotechnology and photobiotechnology,” Eng. Life Sci., Nov. 2018. T. Krieg, J. Madjarov, L. F. M. Rosa, F. Enzmann, F. Harnisch, D. Holtmann, K. Rabaey, „Reactors for Microbial Electrobiotechnology,” In Advances in biochemical engineering/biotechnology (pp. 127–141), 2018. F. Enzmann, F. Mayer, M. Rother, D. Holtmann, “Methanogens: biochemical background and biotechnological applications,” AMB Express, vol. 8, no. 1, p. 1, 2018. Further Publications F. Mayer, F. Enzmann, A. M. Lopez, and D. Holtmann, “Die mikrobielle Elektrosynthese von Methan,” BIOspektrum, vol. 23, no. 4, pp. 471–473, 2017. Publications and Presentations III Poster Presentations F. Mayer, F. Enzmann, A. Martinez-Lopez, D. Holtmann: Methanogens for Microbial Electrosynthesis; Himmelfahrtstagung 2017 - Models for Developing and Optimizing Biotech Production, Neu-Ulm, 22.-24.5.2017 F. Enzmann, F. Mayer, D. Holtmann: Reactor design for bioelectromethanogenesis; Ismet 6, Lissabon, 3.-6.10.2017 F. Enzmann, D. Holtmann: The BIG challenge - Using Similarity Theory for the Scale Up of BES; 4th EU-ISMET , Newcastle upon Tyne, 12.-14.9.2018 F. Enzmann, F. Mayer, D. Holtmann: Transferring bioelectrochemical processes from H-cell to scalable reactor concepts – Bioelectrochemical bubble column; Himmelfahrtstagung 2018 - Heterogeneities - A key for understanding and upscaling of bioprocesses in up- and downstream, Magdeburg, 7.-9.5.2018 Conference Talks F. Enzmann: Scalable reactors for the bioelectrochemical conversion of CO2 to CH4; Nachwuchsforscherkongress „24 Stunden für Ressourceneffizienz“ 2018, Pforzheim, 27./28.2.2018 F. Enzmann: We do it our way – Influences on the mechanism of electron uptake in methanogens; 4th EU-ISMET , Newcastle upon Tyne, 12.-14.9.2018 F. Enzmann: Scale Up of Bioelectrochemical reactors; Himmelfahrtstagung 2019: Intensification and digitalisation for integral bioprocessing, Hamburg, 25.5-28.5.2019 Abstract IV Abstract An upcoming technology in the field of biological research is bioelectrochemistry, which combines electrical energy and biological processes. The proof of concept for several bioelectrochemical processes was shown in lab-scale applications, and a variety of different bioelectrochemical systems were constructed. Since no common characterization strategies were applied to these reactors and no consensual performance parameters were used to show the results of the processes, comparisons are difficult. This was identified as a major drawback on the way to an industrial application of bioelectrochemistry, since rational optimizations are hardly possible. Only few processes were transferred to bigger scales so far, and long term operations under industrial conditions are rare. The objective of this doctoral thesis was the design, characterization and Scale-Up of a bioelectrochemical system. The main application for this reactor was bioelectromethanogenesis, which was supposed to be continuously optimized during the project. In this technique, anaerobic methanogens are fed with electrons supplied by a cathode and CO2, and convert these substrates to methane. Since fossil fuels are about to be depleted during the next decades, methane could be a substitute fuel, base chemical or substrate for further biological conversions. A bioelectrochemical system with a bubble column working chamber was designed. The working volume of the device was one liter, with a surrounding counter chamber of 10 liters. The working electrode was placed in the center of the reactor, while the counter electrode was wrapped around the working chamber. The reactor was abiotically characterized using various methods, such as cyclic voltammetry, chronoamperometry and kLa measurements to allow comparability to other set-ups. It could be shown by calculation of the Wagner number, that the electrical field within the system was evenly distributed and by calculation of the Reynolds, Bond and Weber number that a laminar, homogeneous, bubbly flow established itself in the working chamber. By continuous gassing with oxygen-free in-gas streams, it was possible to host anaerobic methanogenic archaea, to provide them with gaseous CO2 contained in the in-gas and electrons from the working electrode as substrates and to produce methane. A pure culture of Methanococcus maripaludis was used as biocatalyst to allow comparison to published research data. The first setup already showed a methane production of 0.23 mmol*d-1, which equaled a specific methane production rate of 33.8 mmol*d-1*m-² at a Coulombic efficiency of 51.0 %. This was higher than reported for bioelectromethanogenesis with M. maripaludis before. The space-time-yield was comparable to commonly used H-cell reactors tested for comparison, which contain a working volume of 100 ml. The process of bioelectromethanogenesis was further optimized, leading to a 9.8 fold increase in methane production rate to 2.3 mmol*d-1 at a Coulombic efficiency of 56.4 %. The specific methane production rate in this case was 81.4 mmol*d-1*m-², which was also comparable to data shown for bioelectromethanogenesis with mixed cultures, which do usually result in higher production Abstract V rates. The results obtained with the bubble column reactor were also comparable to those obtained in a bioelectrochemical stirred tank reactor with similar operating conditions and better than in H-cells under similar conditions, including same electrode material, membrane material and working potential. The most efficient step of optimization was the shift of the applied working potential from -0.9 V vs. Ag/AgCl to -1.1 V vs. Ag/AgCl, which caused a 143.2 % higher total methane production rate, followed by the quadrupling of the electrode surface area, leading to a 139.6 % improvement of the total methane production rate, although the specific methane production rate based on the geometrical surface area declined. The change of the electrode type caused a 54.3 % improvement. The change of the CO2 content in the in-gas from 20 % to 100 % was increasing the total methane production rate by 83.9 %. The increase of the gassing rate from 30 ml*min-1 to 90 ml*min-1 improved the total methane production rate by 35.8 %. Taking the various influences of the different optimizations into account, it seems likely that the electron availability is the main limitation of the process, followed by the CO2 availability. No significant improvement of the process was observed when adding 3-(N-morpholino)- propanesulfonic acid buffer to the medium, when increasing the anode area or when changing the initial optical density of the microorganisms. The biological aspect of the system is therefore not