Titel:			Dissertation Melanie Tabea Knoll - Appendix 6.4: Detachment of biomaterial - OCT and video time lapse
DOI of dataset: 	https://doi.org/10.15480/882.8701
First Author: 		Melanie Tabea Knoll, melanie.knoll@tuhh.de, Technische Mikrobiologie V-7, ORCiD: 0000-0003-1009-3055
Corresponding author: 	Johannes Gescher, johannes.gescher@tuhh.de, Technische Mikrobiologie V-7, ORCiD: 0000-0002-1625-8810

Description of the research project:

The ongoing climate crisis highlights the need to rethink the way humanity consumes and produces resources. Bioelectrochemical systems (BES) offer the possibility of producing sustainable electricity. 
In these systems, electroactive microorganisms catalyze the conversion of chemical into electrical energy and vice versa.The microorganisms can utilize biological waste streams as substrate and transfer
their respiratory electrons on the BES electrode, generating electricity in a sustainable manner. To facilitate this electron transfer, the organisms colonize the electrode surface in the form of a biofilm. 
The biofilm-electrode interaction is a key factor that can limit sufficient space-time-yield required for industrial applications.Providing the organisms with an artificial scaffold that enhances this 
interaction compared to the naturally formed biofilm matrix can significantly improve current production. In this work, such a hybrid biomaterial was established by embedding the electroactive model organism 
Shewanella oneidensis in an agarose hydrogel. The possibility of detaching the hybrid biofilm material should be investigated, as the recovery of the biomaterial could be essential in future application processes
and reveal the maximum number of electrons that can be supplied before the material degrades. The latter is an important parameter for the potential application of the biomaterial in bioelectrosynthesis, a 
process in which organisms grow on the cathode as a source of electrons and energy and where the biomaterial could have similar beneficial effects on productivity. 


Method / Description of data:
Synthetic biofilm detachment was induced by applying negative current to a sprayed hydrogel to the BES flow cell reactor. For this, 5 mL of a 1.8 % agarose hydrogel was sprayed once onto graphite felt and 
once onto a graphite plate. After not more than 1 min, BES media without lactate was poured on top of the hydrogel to prevent dehydration. The flow cell was closed with the polycarbonate window and metal 
top and the system was completely filled with media (~ 50 mL). The flow cell was mounted horizontally onto a metal rack, allowing for the analysis via optical coherence tomography (OCT) as shown by 
Hackbarth et al. (2020). OCT is a non-invasive imaging method that allows the visualization of 3D structures including biofilms. Thereby, a light beam is reflected and scattered by the sample to be observed
and subsequently interferes with the light beam of a reference (Wagner and Horn, 2017). This interference is captured by a detector and analysed to obtain a three-dimensional representation of the sample. 
Since the light is able to penetrate the sample to a certain degree, a depth profile can be created at each scanned point (A-scan in Z-direction). If such a depth profile is then recorded along a defined axis,
a 2D view of the biofilm is obtained (B-scan in XZ-direction). Many such two-dimensional "lines", consecutively one after the other, finally result in a 3D image of the biofilm (C-scan in XYZ direction; 
Wagner and Horn, 2017). To visualize the detachment, OCT C-scans were used for a before and after picture of the 3D structure of the agarose hydrogel. Further, B-scans were filmed using the Windows Xbox Game
Bar (version 5.823.1271.0) to give a 2D view of the detachment process. The OCT device (Ganymede II – LSM04, Thorlabs, Dauau, Germany) was therefore mounted onto the metal rack to visualize the back part of 
the BES flow cell. As the detachment process was visible without optical magnification as well, a video recording of the hydrogel was made using a mobile phone camera (Samsung S20 FE, version 13.1.00.50, 
Samsung Electronics, Suwon, South Korea). Therefore, the mobile phone was fixed onto the metal rack to visualize the front part of the BES flow cell. The videos of the OCT images and the mobile phone camera 
were then displayed side by side and compared for the different applied currents. For video processing the Microsoft video software (version 2023.10030.7003.0) and the AnyMP4 video converter ultimate 
(version 8.5.20) were used. 

Context / Results / Conclusions:
In conclusion, the graphite felt resulted in partial detachment of parts of the biofilm, whereas the graphite plate resulted in complete detachment of the biomaterial as a whole. This difference might be
attributed to the nature of the materials themselves. A graphite plate is a planar electrode material with a defined surface area, while graphite felt offers a three-dimensional specific surface area that 
is described as being more favorable for the formation of electroactive biofilms (Guo et al., 2015). The fibrous nature of the graphite felt might facilitate H2 formation more abundant over the complex 
surface area compared to the graphite plate, resulting in the observed partial rather than the total detachment.
For application, the detachment experiment can provide another insight into the maximum number of electrons that can be supplied to a cathodic biofilm before the biomaterial would start to degrade. The 
formation of H2 bubbles was visualized to begin at an applied current of -2 mA cm^-2, defining the maximum current that can be applied before biomaterial degradation occurred. From this value, the number 
of electrons can be calculated on the basis of the relation between amperes and coulombs, as follows. The application of one ampere is equivalent to the delivery of one coulomb per second, and one coulomb
is defined as the delivery of 6.24 x 10^18 electrons. This gives a maximum of 1.25 x 10^20 electrons per second per square meter of electrode surface that can be supplied in this system before biomaterial 
degradation occurs. Using Avogadro's constant (NA; 6.02 x 10^23 mol^-1), this refers to 0.75 mol electrons per h and per square meter electrode surface (mol h^-1 m^-2). Based on this, the theoretical maximum 
amount of, for example, ethanol or biomass that can be produced from the CO2 fixation of electrotrophic organisms in MESC systems can be calculated. Thereby, the number of electrons required to reduce CO2 
determines the productivity of these production processes. This can be derived from the oxidation state of the carbon in the individual molecules. For CO2 and ethanol (C2H6O) the oxidation state can be 
deduced from the sum formula to be -IV and +II respectively, whereas in cellular biomass the oxidation state of carbon is reported to be approximately ±0 (Dick, 2014). Thus, 12 electrons are required to 
produce 1 mol ethanol, which is formed from 2 mol CO2. Given the maximum electron supply of 0.75 mol h^-1 m^-2, this results in a maximum productivity of 62 mmol ethanol h-1 m-2 that could be produced by 
the applied biomaterial in MESC processes. Biomass production, on the other hand, requires approximately 4 electrons to reduce 1 mol CO2 (Dick, 2014), corresponding to a maximum production of 187 mmol 
biomass h^-1 m^-2. Accordingly, the theoretical maximum given here is an indication of the extent to which the biomaterial could support the improvement of ethanol production before hydrogel degradation occurs.




References:
Hackbarth, M., Jung, T., Reiner, J.E., Gescher, J., Horn, H., Hille-Reichel, A., and Wagner, M. (2020) Monitoring and quantification of bioelectrochemical Kyrpidia spormannii biofilm development in a novel flow cell setup. Chemical Engineering Journal 390: 124604.
Wagner, M., and Horn, H. (2017) Optical coherence tomography in biofilm research: A comprehensive review. Biotechnology and Bioengineering 114 (7): 1386–1402.
Guo, K., Prévoteau, A., Patil, S.A., and Rabaey, K. (2015) Engineering electrodes for microbial electrocatalysis. Current Opinion in Biotechnology 33: 149–156.
Dick, J.M. (2014) Average oxidation state of carbon in proteins. Journal of the Royal Society Interface 11 (100): 20131095.


Excerpts from the dissertation "Sprayable biofilm - Agarose hydrogel as 3D matrix for enhanced productivity in bioelectrochemical systems" by Melanie Tabea Knoll