Materials：9k-S0 plates, LB plates, Starkey-Na2S203 solid medium,1.5 mL centrifuge tube, PBS buffer, 9k buffer
1.Collection of Bacterial Cells:Acidithiobacillus ferrooxidans ATCC 23270, serving as the recipient strain, was cultivated in 9K-S0 medium for three days until it reached the logarithmic growth phase. The culture contained 1% (w/v) elemental sulfur, and the cells were collected by centrifugation at 6000 rpm for 3 minutes when the OD600 reached 0.4. E. coli SM10, carrying the recombinant plasmid and acting as the donor strain, was subcultured from an overnight culture into LB medium supplemented with 100 μg/mL streptomycin and grown to an OD600 of 0.6 to 0.8. The cells were then collected by centrifugation at 7000 rpm for 3 minutes.
2.Washing of Bacterial Cells: The recipient cells were washed with a 2:2 wash solution and centrifuged at 2000 rpm for 1 minute to remove elemental sulfur. The supernatant was decanted as the cell suspension.
3.Conjugation: Both the recipient and donor cells were concentrated to an OD600 of 1. They were mixed in a 1:2 ratio (recipient:donor), and 100 μL of the mixture was evenly spread onto a 2:2 solid conjugation plate containing a 0.45 μm filter membrane. The plates were incubated at 30℃ for 5 days in an upright position.
4.Membrane Washing: The 0.45μm membrane was removed from the plate and placed into a sterile 50 mL EP tube. The cells on the membrane were washed off with 5 mL of 2:2 wash solution, collected by centrifugation, and concentrated into a 1.5 mL EP tube. Serial dilutions were made at 10-fold, 50-fold, and 100-fold. An aliquot of 100 μL of the concentrated cell suspension was spread onto Starkey-Na2S203 solid medium supplemented with 100 μg/mL streptomycin and incubated at 30℃ for 15-20 days, inverted, until colonies were observed.
2.5.Verification: Single colonies were first inoculated into 10 mL of 9K liquid medium containing 10% Fe2+ (49.64 g/L FeSO4·7H2O) without antibiotics and cultivated for 3 days until the culture turned red. The culture was then transferred into 30 mL of 9K-S0 liquid medium containing 200 μg/mL streptomycin for further cultivation for 6 to 10 days. The sulfur particles were observed to be fine and did not clump together, settling at the bottom with a turbid supernatant upon standing. A 20 mL aliquot of the culture was used to extract plasmid DNA, and PCR was performed for verification. If successful, subsequent experiments were conducted.

Materials：LB plates, 1.5 mL centrifuge tube, 96-well plate, Micro plate reader (produced by gene5®)
1.Preparation of LB Liquid Medium: The engineered E.coli BL21 strain, cultured to the mid-log phase (OD600 ~ 0.5), was used to inoculate fresh LB medium at a 1% inoculation ratio.
2.Cultivation: The culture was allowed to a shaking incubator set at 37 ℃,180 rpm for a period of 2 days. Samples were collected at 0, 5, 10, 15, 20, 25 and 30 hours post-inoculation to monitor the OD changes. Three parallels were set up for the experiment.
3.Sampling Method: Aliquots of 200 μL of the supernatant were taken to measure the OD600. The growth curve was plotted based on the changes in OD600. Each sample was tested in triplicate to ensure accuracy and consistency.

Materials：9k-S0 plates, 9k-Fe2+ plates, 1.5 mL centrifuge tube, 96-well plate,Micro plate reader
1. Preparation of 9k-Fe2+ Liquid Medium: The engineered A. ferrooxidans strain, cultured to the mid-log phase (OD600 ~ 0.5), was used to inoculate fresh 9k-Fe2+ medium at a 1% inoculation ratio.
2. Cultivation: The culture was allowed to a shaking incubator set at 30 ℃ ,180 rpm for a period of 7 days. Samples were collected starting at the time of inoculation, with one sample taken every 12 hours to monitor OD changes. Three parallels were set up for the experiment.
3. Sampling Method: Aliquots of 200 μL of the supernatant were taken to measure the OD600. The growth curve was plotted based on the changes in OD600. Each sample was tested in triplicate to ensure accuracy and consistency.

Materials for 1 L: (NH4)2SO4 30 g
KCl 1 g
K2HPO4 5 g
MgSO4·7H2O 5 g
Ca(NO3)2 0.1 g
Protocol:
1. Measure materials to scale that in need
2.Add the required proportion of water, shake the bottle until the solids completely dissolve.
3. Adjust pH to 2.0 using 30% concentrated sulphuric acid.
4.Place the bottles into the autoclave for sterilization, under 121°C for 30 minutes.
5.Let the bottles cool down under room temperature. The solution would preserve under room temperature for a maximum of 7 days.
6. Add 1% sulphur monomer or 44.22 g/L ferrous sulphate heptahydrate depending on culture conditions

Materials for 1 L: LiquidA： (NH4)2SO4 6 g
KH2PO4 6 g
MgSO4·7H2O 0.1 g
CaCl2 0.37 g
LiquidB:
agar 20 g
LiquidC：
Na2S2O3·5H2O 3 g
FeSO4·7H2O 0.012 g
20 mL H2O
Protocol:
1.Measure materials to scale that in need
2.Add the required proportion of water, shake the bottle until the liquid A, B, C solids completely dissolve.
3.Place the liquid A, B bottles into the autoclave for sterilization, under 121°C for 30 minutes.Liquid C filtrate for sterilization
4. Mix equal volumes of liquid A and liquid B, then add 5% of liquid C of the total volume of liquid A and B, mix well and prepare the medium plate.

Materials for 1 L：Na₂HPO₄: 6.0 g
KH₂PO₄: 3 g
NaCl: 0.5 g
NH₄Cl: 1 g
MgSO₄·7H₂O: 0.246 g
CaCl₂·2H₂O: 0.0146 g
Glucose (C₆H₁₂O₆): 4 g
Protocol:
1.Weigh the reagents in proportion: Prepare a 2× medium according to the above formula, and prepare glucose separately (if the preparation volume is not 1 liter, adjust the reagent amounts proportionally).
2.Add the corresponding proportion of water and shake the bottle until the solid reagents are completely dissolved.
3.Sterilize the bottles in an autoclave at 121°C for 30 minutes. Sterilize glucose separately in an autoclave at 105°C for 45 minutes.
4.Allow the bottles to cool to room temperature. Store the solution at room temperature.

Materials:M9 Liquid Medium,96-well plate,1.5 mL centrifuge tube, Micro plate Reader 1.For the assay, two reagents were needed. Reagent I, the periodate reagent, consisted of 18 mg mL-1 sodium periodate (Merck) dissolved in distilled water with 10% (v/v) acetic acid (Merck). For preparation purposes, sodium periodate was fifirst dissolved in water. After the addition of acetic acid and adequate mixing, 77 mg mL-1 ammonium acetate (VWR) was added. The amount of sodium periodate in this reagent was calculated for a calibration curve from 50 mg L-1_200mgL-1 glycerol. Reagent II, the acetylacetone reagent, was composed of 1% (v/v) acetylacetone (VWR) in isopropyl alcohol (Roth). This reagent had to be stored in the dark.
2.The measurement was executed in a microplate reader (Synergy HT BioTek, Winooski, USA). Therefore, an amount of 40 μL sample (cell-freesupernatant of the fermentation) was pipetted into each well of a standard 96 well plate (Greiner). Then, 40 μL Reagent I was added and mixed adequately. After an incubation time of 10 min, 125 μL Reagent II was pipetted into each well and mixed adequately. The absorption at 410 nm was measured over a period of 25 min in the plate reader.The glycerol content was calculated with Eq , based on a glycerol standard curve (50 mg L-1–200 mg L-1)
3.The assay can also be performed in a cuvette using a spectrophotometer. In this instance, 5-fold the amount of sample and reagents must be used.

1.Medium 9K is composed of Solution S1 and S2, with the following final concentrations of its components: 3.0 g/L (NH₄)₂SO₄, 0.5 g/L MgSO₄·7H₂O, 0.5 g/L K₂HPO₄, 0.1 g/L KCl, 0.01 g/L Ca(NO₃)₂, and FeSO₄·7H₂O (44.22 g/L stock solution, adjustable as needed). Solution S1 contains (NH₄)₂SO₄, MgSO₄·7H₂O, K₂HPO₄, KCl, and Ca(NO₃)₂ dissolved in deionized water. The pH of Solution S1 is adjusted to 2.0 using H₂SO₄ and then autoclaved. Solution S2 consists of FeSO₄·7H₂O dissolved in deionized water, with its pH also adjusted to 2.0 using H₂SO₄, and is sterilized by filtration through a 0.2 μm filter. Under aseptic conditions, Solution S1 and Solution S2 are mixed and aliquoted into clean, screw-cap shake flasks. All electrochemical experiments were conducted using a three-electrode system, comprising a working electrode (which served as the cathode in this context), a counter electrode, and a reference electrode.
2.The three-electrode system was assembled in a sterile reactor: both the cathode (area 1 cm × 3 cm = 3 cm²) and anode (area 3 cm × 4 cm = 12 cm²) were constructed using hydrophobic carbon cloth, with the cathode potential set at -0.2 V vs. Ag/AgCl (saturated KCl, E° = 0.197 V vs. SHE). The reactor consisted of a 100 mL glass vial equipped with a perforated rubber stopper and parafilm sealing (for electrode fixation, allowing gas exchange while preventing contamination). Current collectors made of 1.5 mm titanium wire were connected to the electrodes. The electrolyte was 9K medium (supplemented with ferrous iron, pH = 2). 9 mM sodium citrate (C₆H₅Na₃O₇·2H₂O) was added to reduce Fe³⁺ precipitation and enhance current output.The three-electrode reactor system used in this study is shown in Figure 1, and the laboratory setup of the reactor is presented in Figure 2.
3.A. ferrooxidans was cultured in a 250 mL shake flask containing 150 mL of 9K medium (supplemented with 44.22 g/L FeSO₄·7H₂O) at 30°C and 150 rpm. Cells were harvested by centrifugation at 5500 rpm for 5 minutes and resuspended in fresh medium. The reactor was inoculated with this cell suspension to an initial OD₆₀₀ of 0.1. The electrodes were connected to a potentiostat to record the current-time curves generated by the engineered strain. The reactor was then placed in a constant-temperature incubator at 30°C.

Figure 1 Three-electrode reactor system used in this study for biocathode development.

Figure 2 Electrochemical reactors under laboratory condition.