In extracellular electron uptake (EEU), electrons first be taken up from electrode to OmcA and MtrC, directly or through electron mediators like riboflavin. Then electrons go from MtrABC system to inner membrane cytochrome, CymA, mainly through shutters like the small tetrahaem cytochrome CctA (STC). And finally some electrons reach formate dehydrogenases (FDHs) and reduce CO2 to formate. The following figure shows the pathway of electrosynthesis of formic acid.

To enhance the produce of formate, we consider to optimize the extracellular electron uptake (EEU) pathway.

First, CctA, as a mainly used electron shutter in periplasmic, has been proposed to be the bottleneck of EEU efficiency. Overexpression of the periplasmic electron shutter protein CctA can enhance electrosynthesis efficiency and promotes formic acid production.

We drew the plasmid map according to the literature.

This plasmid can’t differentiate transferred Shewanella from those that don’t have the plasmid, because according to the literature, WT Shewanella is resistant to ampicillin.

Limited by condition and budget, we can’t obtain pBBR1MCS-2, thus we need to find a suitable plasmid to integrate cctA.We need to change the resistant gene.Refer to the initial design, we substitute the AmpR gene for KanR gene.

S.oneidensis MR-1 is Gram-negative and facultative aerobic. Under elevated oxygen concentrations, aggregating can be observed. Firstly, we activate MR-1 with TSA solid media. Then we choose to use LB aerobic culture media to cultivate MR-1 at 35℃, 200pm. and determine the optical density (OD600) every 2 hours to get the growth curve and growth rate of S.oneidensis MR-1. Besides, we use wild type strain to explore the essential conditions for electrosynthesis in S.oneidensis MR-1. We set up three different conditions: no carbon dioxide and no electricity, carbon dioxide flowing but no electricity, and both carbon dioxide flowing and electricity on.

1.We use LB broth to cultivate S.oneidensis MR-1 wild type: LB (Peptone 10g/L, Yeast Extract 5g/L, NaCl 10g/L); After inoculation, OD600 was measured every two hours, and last for 24 hours.
2.When OD600 > 1, we transfer the LB 1 mL culture media inoculated with MR-1 wild type to Shewanella Basal Buffer Solution for S.oneidensis MR-1 (SBBS-o) cathode liquid culture media for culture at 35℃, 4hr.
3.Add electrode carbon cloth into the cathode liquid to continue to culture at 35°C, 200pm for 8-12 h.
4.Assemble the electrosynthesis device, adding 200ml anode electrolyte to anode and cathode electrolyte respectively. Then add 4 mL of lactic acid and 200uL sodium nitrate into the cathode electrolyte. And determine the initial OD600.
5.Connect the device with adjustable regulated DC power and adjust the voltage to 1V. Open the CO2 valve, maintain 60 ml/min air flow rate.
6.Conduct electrosynthesis experiments for 6 hours, measuring OD600 every 2 hours, and using the thiobarbituric acid colorimetric (TBA) method to measure formic acid content.
S.oneidensis MR-1 wild type electrosynthesis condition (N for No and Y for Yes):

Growth Conditions: 200mL LB Broth in 500mL Erlenmeyer flask, 30 oC, 225 rpm
Every two hours we measured optical density (OD600).
Growth Rate (R) = 0.026 generations/min
Generation Time (G) = 1 / R = 38.40 min

According to a literature in 2008[A], we design and reform a thiobarbituric acid colorimetric (TBA) to measure the formic acid content produced by S.oneidensis MR-1. The standard curve has good linearity and the results are reliable.

We set up a series of time gradients to explore the optimal reaction time between thiobarbituric acid and formic acid, which indicates that 20min, 100℃ is the optimal heating time. We found 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. And the measured formic acid content is low. So we need to over express ccta gene to improve the efficiency of formic acid production.

Consistent with Vibrio natriegens, S.oneidensis MR-1 is also transformed by electroporation. We use SOB medium and SOC-s recovery solutions post-electroporation. Detailed procedures can be found in the protocol section for S.oneidensis MR-1 transformation. After transformation, we proceed expression validation. The validation process consists of two steps. The first step is to screen for colonies that have successfully incorporated the plasmid from the resistant plates. The second step is to detect whether the target gene is expressed. However, we encountered difficulties in protein extraction due to the small molecular weight of the Ccta protein. Therefore, we only assessed whether the ability of the engineered S.oneidensis MR-1 to produce formic acid improved after introducing the plasmid as an indicator of target gene expression.

According to the protocol, we conducted multiple transformation experiments with voltages ranging from 0.3 to 0.7 kV. The selected media included SOB (Super Optimal Broth) and SOC-s (Super Optimal Catabolite Repression Broth for SOMR-1). Detailed procedures and results can be found in the protocol. As for expression validation. First, we spread the transformed S.oneidensis MR-1 onto kanamycin-resistant plates and observed for colony formation after one day. We performed multiple transformations, taking about a week in total to obtain colonies that could grow on the resistant plates, indicating that the plasmid was successfully introduced. Subsequently, we conducted electro-synthesis experiments with the engineered strain containing the plasmid. Similarly, we carried out the electro-synthesis experiments for 6 hours, measuring OD600 every 2 hours and using the TBA method to assess formic acid content.

We performed multiple transformations, taking about a week in total to obtain colonies that could grow on the resistant plates, indicating that the plasmid was successfully introduced. 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%.

During the initial transformation, the culture medium was contaminated due to improper operation, and then the transformed bacteria were not the target bacteria. We optimized the experimental treatment environment to contain the electroporation under sterile condition. The final results indicated that the engineered strain with the plasmid produced more formic acid, which provides some evidence for the successful expression of the ccta gene.

The MR-1 after transformation of ccta has been shown to greatly improve its production efficiency of formic acid. At the same time, the electron mediator RF and 2HNQ can also improve the ability of MR-1 to absorb electrons and produce formic acid[B]. Therefore, in order to quantify the ability of the transformed MR-1 to produce formic acid with the assistance of an electron mediators, we set up a series of comparative experiments with MR-1 wild type.

1.We established two liquid culture media to cultivate transformed S.oneidensis MR-1: LB (Peptone 10g/L, YE 5g/L, NaCl 10g/L); TB(Tryptone 12g/L, YE 24g/L, K2HPO4 12.54g/L, KH2PO4 2.313g/L, Glycerol 4ml/L). After inoculation, OD600 was measured every two hours.
2.When OD600 > 1, we transfer the LB media inoculated with transformed MR-1 to the SBBS-o cathode liquid culture media for culture at 35℃, 4hr.
3.Inoculate 2mL of MR-1 LB liquid medium into the cathode culture medium. Add electrode carbon cloth and electronic mediator(Riboflavin, RF; 2-Hydroxy-1, 4-naphoquinone, 2-HNQ) into the cathode liquid to continue to culture at 35°C, 200pm for 8-12 h, rGO is not necessary.
4.Assemble the electrosynthesis device, adding 200ml anode electrolyte and cathode electrolyte respectively. Then add 4 mL of lactic acid and 200uL sodium nitrate into the cathode electrolyte.
5.Connect the device with adjustable regulated DC power and adjust the voltage to 1.1V. Open the CO2 valve, maintain 60 ml/L min air flow rate.
6.Electrosynthesis every for 2 hr, 1000uL, 800uL two-tube samples are taken from the double-chamber electrolytic cell to test the OD600 and formate content.
MR-1 contrast experiment electrosynthesis condition:

MR-1 contrast experiment electrosynthesis condition:

And rGO could interact with RF to improve the capacity of electrosynthesis of formic acid.

We used thiobarbituric acid colorimetric (TBA) to measure formic acid content, obtaining reliable results. Transformed MR-1 with 2HNQ/RF reached a formic acid peak of 7-10 mM in 6 hours, with 2HNQ showing better electron trapping efficiency. While rGO helped MR-1 produce formic acid quickly, it did not significantly increase the peak content.

We found that RF dissolved and appeared yellow, interfering with formic acid measurements. Using powdered activated carbon for decolorization reduced interference, but the formic acid content was lower than expected, likely due to incomplete cleaning of the activated carbon.