Team:Westminster UK/Description
Project
Description and Inspiration
Our society is still largely dependent on humanity’s primal technologies of burning resources for generating energy with fossil fuels. Coal, oil and gas predominantly contribute to global warming emissions (Figure 1) and comprise 80% of our main energy resources. The dependence on non-renewable energy sources continues to rise, along with the main complications being their limited availability and contribution to heavy air pollution, which is detrimental to human health and the environment. Due to their highly polluting properties to the atmosphere and the biosphere, reducing our reliance on burning fossil fuels and finding alternate energy sources is an utmost priority.
Figure 1: Global warming emissions from non-renewable and renewable energy sources
Figure 2: Meet the focus chassis of our work - Shewanella oneidensis
Shewanella (Figure 2) is a facultative anaerobe, meaning that when oxygen is present, it uses the oxygen as an electron acceptor, allowing the bacterium to effectively respire. However, in the absence of oxygen, Shewanella utilises an alternative respiratory pathway called the Mtr pathway (Figure 3), which enables it to use extracellular elements to reduce its freely flowing electrons.
Although, the pathway is not completely understood yet, there is evidence on the presence of electroconductive nanowires that are widely present during the phases of oxygen depletion (Subramanian et al, 2018). It was found that this pathway works along the double layered cell membrane structure of Shewanella, with much of its outer components being electro-active under conductivity tests of the nanowires and characteristically spiking outside of their cell bodies (Figure 2).
Figure 3: The proposed mechanism of the Mtr pathway
With this in mind, it was found that the conductive properties of these bacteria can in fact be utilised, by the implementation of a method using microbial fuel cells (MFCs)(Figure 4).
Figure 4: The mechanism of an MFC, working much as a galvanic cell, with bacteria cultured directly on the anode of the anode chamber. Reduction of the anode causes an electric current to flow through an external wire connecting the anode with the cathode - making the produced electric energy harvestable.
The main goal of our laboratory work was to characterise the activity of this pathway in Shewanella and contribute with our new findings to further research in understanding how to improve the efficiency of this otherwise fairly inefficient process.
While synthesising the whole pathway, the main focus of our project was on indirect methods of boosting the cells’ exoelectrogenic activity and we found that this can be achieved through multiple different paths. One of these involves understanding how the bacteria respond to low oxygen conditions - it was found that under low oxygen conditions, higher presence of nanowires was identified, while the electric conduction drastically improved, indicating that a metabolic switch stimulated the bacteria into expressing more Mtr components (Barchinger et al, 2016). Consequently, multiple transcription factors were identified to modulate this expression: CRP, EtrA and ApoE.
We found that along with these, specific exo-electrogenic bacteria such as Pseudomonas aeruginosa were tested with the overexpression of RhlA genes, which were found to be responsible for rhamnolipid surfactant production. Specifically, it supplies the acyl moieties for rhamnolipid biosynthesis by competing with the enzymes of the type II fatty acid synthse (FASII) cycle for the beta-hydroxyacyl-acyl carried protein (ACP) pathway intermediates. This process ultimately allows a higher permeability of the cell membranes, allowing further electrically charged flavins and cytochromes to leave cells and bind onto an anode. Since this pathway was not well identified in Shewanella, we designed genetic vectors for it with the hopes of finding similar effects in our tests.
Plasticity
Since Shewanellas are known to be highly versatile when it comes to their digestion preferences, one application for MFCs we are passionate about is the utilisation of plastic biodegradation as the first step to the electric conduction process.
To do this and have as complete of a model for this integration, we collaborated with other iGEM teams that have a focal point on plastic degradation. Since the complete models would ultimately depend on a large number of variables, our model was focused on the foundations of co-culturing that would be used and degradation enzymes / electron shuttling cytochromes involved, with a focus on PET polymers. For the first time, we included an overview of the implementation of eukaryotic organisms (obtained through collaboration) as primary degradation stage units, as well as defining the complete pathway involved in the degradation of PET polymers along with the natural PET degrading organism, Ideonella sakaiensis.
Head bibliography:
Barchinger, S. E., Pirbadian, S., Sambles, C., Baker, C. S., Leung, K. M., Burroughs, N. J., Golbeck, J. H. (2016). Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation. Applied and environmental microbiology, 82 (17), 5428–5443. doi:10.1128/AEM.01615-16
Breuer, M., Rosso, K. M., Blumberger, J., Butt, J. N. (2015). Multi-haem cytochromes in Shewanella oneidensis MR-1: structures, functions and opportunities. Journal of the Royal Society Interface.
Fapetu, S. A., Keshavarz, T., Clements, M. O., Kyazze, G. (2017). Overexpression of the Mtr pathway in Shewanella oneidensis for bioelectricity production, Society for Applied Microbiology.
Gomaa, O. M., Fapetu, S., Kyazze, G., Keshavarz, T. (2018). Probing the mechanism of simultaneous bioenergy production and biodegradation process of Congo red in microbial fuel cells. Journal of Chemical Technology & Biotechnology.
Kouzma, A., Kasai, T., Hirose, A., Watanabe, K. (2015). Catabolic and regulatory systems in Shewanella oneidensis MR-1 involved in electricity generation in microbial fuel cells, Frontiers in Microbiology.
Shi, L. Et al (2006). Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1. Journal of Bacteriology.
Subramanian, P., Pirbadian, S., El-Naggar, M. Y. And Jensen, G. J. (2018). Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography. PNAS, 115 (14), 3245-3255.
Zheng, T., Xu, Y. S., Yong, X. Y., Li, B., Yin, D., Cheng, Q. W., Yuan, H. R., Yong, Y. C. (2015). Endogenously enhanced biosurfactant production promotes electricity generation from microbial fuel cells. Bioresour Technol, 197, 416-21.
