Two sets of growth curves were obtained.

Group	Temperature	Illumination
1	Room temperature	Natural illumination
2	25℃	6000lux

Figure 1: Growth curve of cyanobacteria cultured under room temperature and natural light conditions.

Table 2: Growth curve of cyanobacteria cultured in a light incubator.

Colony:

Figure 1. Colony of Shewanella oneidensis> MR-1 on TSA plate

Growth Conditions: 200mL LB Broth in 500mL Erlenmeyer flask, 30 oC, 225 rpm Every two hours we measured optical density (OD600).

Figure 2. S. oneidensis MR-1 Growth Curve

Growth Rate (R) = 0.026 generations/min

Generation Time (G) = 1 / R = 38.40 min

Exploring the Essential Conditions for Electrosynthesis in Shewanella oneidensis MR-1

The wild type strain was used in this experiment. The different conditions set were whether electricity was applied (if applied, then at 1V) and whether carbon dioxide was introduced (if introduced, the flow rate was controlled at 60 mL/min), while other conditions remained the same. The formate concentration in the device was measured every 2 hours.

Group No.	Strain	Initial OD600	Electricity Applied	C02 Flow	Eletron mediator
1	WT	0.812	N	N	N
2	WT	0.812	N	Y	N
3	WT	0.812	Y	Y	N

Figure 3. Electrosynthesis of formic acid in S. oneidensis MR-1 is activated by electricity.

According to data, the group with both electricity and CO2 flow applied showed a surge in the amount of formic acid produced, while the one with only CO2 flow added didn’t change evidently compared to the group without any treatment. It demonstrated that the electrosynthesis system is activated by exogenous electricity instead of CO2, which is consistent with the fact we have already known that this system is the reverse of EET pathway, and electrons are required to drive the synthesis. On the contrary, CO2 itself didn’t affect this process possibly due to low activity of electrosynthesis system. At 6h, the concentration of formic acid lowers, probably because of the effect of massive bacteria colony, leading to a total consumption. From this result we know that electricity is necessary for efficient electrosynthesis of Formic Acid. To produce as much as possible formic acid, CO2, the raw material, should also be added.

Group No.	Strain	Initial OD600	Electricity Applied	C02 Flow	Eletron mediator
...	...	...	...	...	...
4	Engineered Strain	0.731	Y	Y	N

Figure 4. Engineered Strain possessed a higher production of formate.

We imported a plasmid containing ccta gene in order to increase its overexpression, therefore contributing to a higher rate of electrosynthesis, whose principle is explained in design. After the transformation, the concentration of formic acid produced by engineered strain is higher than that by WT strain, with the peak difference about 2mM, increased by 23.4%. Note that the original OD of engineered strain was slightly lower than WT, so this increase could be higher. This result indicates that our design is feasible and effective to rise the electrosynthesis rate.

Compare the Effects of Different Electron Mediators

Group No.	Strain	Initial OD600	Electricity Applied	C02 Flow	Eletron mediator
...	...	...	...	...	...
5	WT	0.766	Y	Y	2-HNQ
6	Engineered Strain	0.752	Y	Y	2-HNQ
7	WT	0.766	Y	Y	RF
8	Engineered Strain	0.752	Y	Y	RF
9	WT	0.784	Y	Y	RF+rGO
10	Engineered Strain	0.773	Y	Y	RF+rGO

Figure 5. Electron mediators have effects on electrosynthesis. The production of formic acid after adding electron mediators to (A) WT or (B) engineered strain. The comparison between WT and engineered strain is visualized in (C).

To explore the effects of electron mediators on the electrosynthesis of formic acid, we tested 2-HNQ, RF and RF+rGO in both engineered and WT strain. In both strains, the trend showed a similar pattern: all the mediators improved the amount of formic acid synthesized, which is more obviously shown in WT strain (about 25% increase in average, while in engineered strain, only a 6% increase is observe). The increase is definitely caused by a mediated, faster electron transport. Only subtle effect on engineered strain is likely because electron transfer is not a limiting factor any more thanks to overexpression of ccta. 2-HNQ had smallest influence on the concentration of formate in both strains. RF+rGO reached a highest increase for WT (33.7%), while this effect is lower than RF in engineered strain (RF+rGO 5.8%, only RF 7.3%). Overall, engineered strain were still capable of more active electrosynthesis, which proved our successful design again. As a result, it is advisable to use electron mediators, especially RF or RF+rGO to increase electrosynthesis.

Designed four media formulations for cultivating V.N. OD measured every two hours

Analysis: High salinity in the medium, replaced with a different medium based on literature research.

We designed three groups of M9 minimal media with different concentrations of sodium formate as the carbon source and sucrose as the auxiliary carbon source to culture Vibrio natriegens. As shown in the figure, the results indicate that:

1. Vibrio natriegens can utilize sodium formate as a carbon source for survival.
2. in the groups with sucrose as an auxiliary carbon source, Vibrio natriegens exhibited rapid growth.

Fluorescence Detection

Upon excitation with red light under the fluorescence microscope, the Vibrio natriegens transformed with the mCherry gene exhibited a stable red fluorescent signal, confirming the successful expression of the CA plasmid.

Through fluorescence microscopy, the detection of Vibrio natriegens samples expressing EGFP showed a significant green fluorescent signal under appropriate excitation at around 488 nm.The observed green fluorescence confirms the successful transformation and expression of the CARPs plasmid, demonstrating that the exogenous gene was effectively transcribed and translated in Vibrio natriegens.