Non-photosynthetic bacteria produce photocurrent mediated by NADH

In recent years, the concern from the global climate change has driven an urgent need to develop clean energy technologies that do not involve combustion process that emit carbon into the atmosphere. A promising concept is microbial fuel cells that utilize bacteria as electron donors in a bio-electrochemical cell performing a direct electron transfer via conductive protein complexes or by secretion of redox active metabolites such as quinone or phenazine derivatives. In the case of photosynthetic bacteria (cyanobacteria) electrons can also be extracted from the photosynthetic pathway mediated mostly by NADH and NADPH. In this work, we show for the first time that the intact non-photosynthetic bacteria Escherichia coli can produce photocurrent that is enhanced upon addition of an exogenous electron mediator. Furthermore, we apply 2D-fluorescence measurement to show that NADH is released from the bacterial cells, which may apply as a native electron mediator in microbial fuel cells.


Introduction
In recent years, energy innovations are directed toward the development of clean technologies that do not involve combustion processes and in this way can reduce carbon emission. A promising energy solution that is extensively developed is microbial fuel cells (MFCs) 1 . The main approach of MFCs is to exploit the ability of bacterial cells to donate electrons at the anode 2,3 of a bio-electrochemical cell. Alternatively, several species can also apply as electron acceptors at the cathode 2,4,5 . The bacterial cells perform external electron transport by 2 kinds of mechanisms: direct and mediated electron transfer. Direct electron transfer (DET) is conducted by metal respiratory (MTR) complexes in the cellular membrane [6][7][8][9] . Mediated electron transfer is performed by native secretion of redox active molecules. Among these molecules are various derivatives of quinones and phenazines [10][11][12][13][14][15] .
The electrical current production can be enhanced by addition of artificial exogenous electron mediators such as cystine, neutral red, thionin, sulphides, ferric chelated complexes, quinones, phenazines, and humic acids [16][17][18][19][20][21] . Among the most efficient bacterial species in MFCs are Shewanella oneidensis and Geobacter sulfurreducens which consist of a relatively high amount of pili and cytochrome types that are capable of charge transfer [6][7][8][9]22,23 . MFCs are not limited to bacteria and can also utilize yeasts 24 . A unique class of MFCs is the biophotoelectrochemical cells (BPECs) [25][26][27][28][29][30] . This approach utilize the ability of photosynthetic organisms to perform external electron transfer, while the source of the electrons originate from both the respiratory and photosynthetic pathways 31 . The major electron mediators in BPECs are NADH and NADPH that can cycle electrons from photosystem I inside the cells and the external anode of the BPEC 25 . Enhancement of the photocurrent can be achieved by the exogenously adding natural electron mediators such as NADH, NADPH and vitamin b1 or the non-natural mediator potassium ferricyanide 30 .
In this work, we show for the first time that it is possible to produce photocurrent from nonphotosynthetic bacteria in a BPEC while the electron transfer is being mediated by NADH.

E.coli releases NADH and FAD to the external cellular medium
Recent works have reported the release of the redox active molecules Nicotinamide adenine dinucleotide (NADH) and Nicotinamide adenine dinucleotide phosphate (NADPH) from various organisms such as cyanobacteria 25,30 , microalgae 26 , seaweeds 32 , plant's leaves 29,34,36 , roots 38 and sea anemones 39 . These molecules can apply as electron mediators in bio-. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 19, 2023. ; https://doi.org/10.1101/2023.01.16.524302 doi: bioRxiv preprint electrochemical cells catalysing electrons transport between the respiration and photosynthetic pathways of the cells and the external anode. The biological reason for the release of NADH and NADPH is not fully understood. It was suggested that it may derive from a minor leak of cytoplasmatic content, be involved in quorum sensing or apply to reduce iron to internalise it 25 .
Mediated electron transport in non-photosynthetic bacteria in microbial fuel cells (MFCs) was previously explained by secretion of quinone and phenazines derivatives [16][17][18][19][20][21] . Nevertheless, the possibility of a release of NADH and NADPH from non-photosynthetic bacteria was not studied yet. To investigate this, we wished to analyse the external cellular solution of E.coli As E.coli not a photoautotroph and cannot produce NADPH in a photosynthetic pathway like cyanobacteria, we suggest that the peaks with maxima at (λex = 300 nm, λex = 450 nm) and (λex = 360 nm, λex = 450 nm) originate from the fluorescence of NAD + and NADH respectively.
Based on the obtained results, we suggest that the identified redox active species NADH and FAD may apply as native electron mediators in MFCs in addition to quinones and phenazines. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  ( Fig. 2a,b)The light intensity at the height of the electrode surface was 1 Sun (1000W at potential varied from Cyclic voltammetry of was measured in dark and light . ) 2 / m 0 to 1 V, with a scan rate of 0.1 V/s. The voltammogram showed 2 peaks at maximal potentials of 0.6 and 0.9 V that were enhanced under illumination (Fig. 2c). The bigger peak around 0.9 V correlates with the voltammogramic fingerprint of NADH , strengthening our hypothesis that this molecules plays a key role in the electron 41 mediation between the bacterial cells and the anode.
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E.coli produces photocurrent in a bio-electrochemical cell.
Next, we wished to explore whether E.coli can produce photocurrent in a bio-electrochemical cell. Chronoamperometry of E.coli was measured with dark/light irradiation intervals of 100 s.
A potential bias of 0.9 V was applied to the WE (Fig. 3). This potential was chosen based on the bigger peak that was obtained in the cyclic voltammetry measurements (Fig. 2). A current of about 0.07 µA /cm 2 was obtained in dark. Upon irradiation, the current was enhanced by ~ 0.05 µA /cm 2 . PBS applied as a control, showing no significant dark current and a photocurrent of ~ 0.005 µA /cm 2 that was obtained because of a direct light absorption in the WE. We postulated that this effect was significantly smaller for the bacterial suspension as their turbidity do not allow the same transparency as the clear PBS solution. Previous studies about photocurrent production from various intact cyanobacterial species, showed a photocurrent of about 0.3 µA /cm 2 (without being normalised to their chlorophyll content), while about 25 % of the current (~ 0.075 µA /cm 2 ) was reported to originate from NADH. A similar photocurrent production was obtained for E.coli (Fig. 3). Based on this, we suggest that in cyanobacterial based BPECs part of the photocurrent does not originate from photosynthesis but from and enhanced formation of NADH catalysed by light irradiation.
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The electron transfer mechanism in photo microbial fuel cells.
Based on the results obtained in this study, and previous studies we suggest mechanisms for the external transfer between the bacterial cells and the anode. In dark and upon association of the bacteria with the active electrochemical cells. NADH molecules are released from the cells donating electrons at the anode to produce electrical current. The oxidized form NAD + is internalized into the cells cytoplasm were it may be re-reduced to NADH in the Glycolysis pathway. Upon irradiation, the reduction of NAD + to NADH is being enhanced, increasing the number of molecules in the NADH cytoplasmatic pools, and the release of these molecules from the bacterial cells to produce electrical current. FADH2 may also be released from the cells to be oxidized at the anode into FAD, that can be reduced again by MTR complexes.
These MTR complexes can also conduct a DET to donate electrons at the anode. DET may also be performed by conductive pili. Some of the non-light dependant electrical current may also derive from secretion of quinone and Phenazine molecules.
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Conclusions
In this work, we showed that non-photosynthetic bacteria can produce photocurrent. We applied 2D-fluorescence spectra of the external solution and cyclic voltammetry of the bacterial cells to show for the first time that some of the electrical current generation originate from NADH. These discoveries may help to improve the current production in MFCs.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 19, 2023. ; https://doi.org/10.1101/2023.01.16.524302 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 19, 2023. ; https://doi.org/10.1101/2023.01.16.524302 doi: bioRxiv preprint