Okkyoung Choi, Taeyeon Kim, Han Min Woo, and Youngsoon Um*

SUPPLEMENTAL MATERIAL

Electricity-driven metabolic shift through direct electron uptake by electroactive heterotroph Clostridium pasteurianum

Okkyoung Choi, Taeyeon Kim, Han Min Woo, and Youngsoon Um*

Affiliation:

Clean Energy Research Center, National Agenda Research Division, Korea Institute of Science and Technology (KIST), Seongbuk-gu, Seoul 136-791, South Korea

*Corresponding Author:

Dr. Youngsoon Um, Clean Energy Research Center, National Agenda Research Division, Korea Institute of Science and Technology (KIST), Seongbuk-gu, Seoul 136-791, South Korea

Tel.: +82-2-958-5819, Fax: +82-2-958-5209, e-mail:

Number of pages: 12

Number of figures: 5

Number of tables: 6

Supplementary Table 1 │Net NADH balance per one mole of product formation from the corresponding mole of glucose and glycerol

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Supplementary Table 2 │The electron and carbon balance. Values for electron and carbon equivalents were calculated from 11 experiment replicates. The values in parentheses for source and product are % of total input and recovered % of total input, respectively.

1Each electron equivalent was calculated by multiplying e- equivalent number per mol by mol of each chemical. Each e- equivalent number per mol is shown as a half reaction in Supplementary Table 3.

2Calculated from consumed glucose concentration

3Electrons consumed by microbes was calculated using with I, the current (A) and F, the Faraday constant (96,485 C/mol e-)

4Chemical formula of biomass is C5H7O2N (8 g COD biomass = 1 e- equiv biomass)1.

5meq H2 = mL H2 × (1 mmol H2/22.4mL) × (273.15 K/310.15 K) × (2 meq e-/mmol H2)

6((e-glucose+ electricity in - e-total recovered)/e-glucose+ electricity in) × 100 in which e-total recovered is the sum of e- equivalents for butanol, acetate, butyrate, biomass, consumed glucose, and H2. For carbon balance, ((Cglucose in - Ctotal recovered)/Cglucose in) × 100 in which Ctotal recovered is the sum of carbon equivalents for butanol, acetate, butyrate, biomass, consumed glucose, and CO2. Lost 14 ~ 20 % in balance analysis might be caused by to gas leaking or omitting of biofilm biomass in calculation.

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Supplementary Table 3│ Half reactions used for establishing electron-equivalent balance and stoichiometry

Supplementary Table 4 │The yield change of products according to current increase with repeated batches

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Supplementary Table 5 │ NADH generation and NADH consumption in BES and control with glucose fermentation. The value was calculated from the average value of replicates.

1NADH generation from glucose= 2Δglucose on the basis of glucose consumption (2 NADH/glucose) (refer to the equation (3) in Methods for calculation).

2, as e- equivalent of NADH is 2.

3NADH consumption = nΔP: ‘n’ is the respective number of NADH requirement per mole of each metabolite (4, 2, and 0 for butanol, butyrate, and acetate, respectively); ΔP (mmol) is the production of each final metabolite (refer to equation (2) in Methods for calculation).

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Supplementary Table 6 │NADH generation and NADH consumption in BES and control with glycerol fermentation (BES: at the condition of 9 mA current consumption)

1, because the glycerol to 1, 3-PD pathway does not involve NADH generation (refer to the equation (3) in Methods for calculation).

2, as e- equivalent of NADH is 2.

3NADH consumption = nΔP: ‘n’ is the respective number of NADH requirement per mole of each metabolite (4, 2, 1, and 0 for butanol, butyrate, 1,3-PD and acetate, respectively); ΔP (mmol) is the production of each final metabolite (refer to equation (2) in Methods for calculation).

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Supplementary Figure 1│The NADH consumption pathway in C. pasteurianum enhanced by the electricity (a) on glucose fermentation and(b) on glycerol fermentation. Bold-squared products increased but dotted squared products decrease in BES.

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Supplementary Figure 2. Current consumption profiles at the first and second batches. The current did not decrease at the second batch right after the suspended culture was drained and fresh medium was added at 60 hr (indicated by the arrow). Because the biocathode remained in the 2nd batch, it shows the direct electron transfer from cathode to biofilm without excreted electron mediators. The surface area of electrode was 108 cm2.

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Supplementary Figure 3│The confocal z-stack images on graphite felt cathode (a) with electricity (200× magnification with 256 x 256 pixel) and (b) without electricity (100× magnification with 256 x 256 pixel) after 24 hr incubation. Graphite felt poised at -0.16 V vs. Ag/AgCl (+0.045 V vs. SHE) shows a well-formed live biofilm along the graphite felt fiber configuration, in comparison to without electricity. Green fluorescence represents active bacteria and red indicates inactive cells (yellow overlap). * Images at ~110 m depth from surface.

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Supplementary Figure 4 │ The scanning electron microscopy (SEM) images of the graphite felt cathode surfaces poised at +0.045 V vs. SHE. White arrow indicates short and thin filamentous appendages and yellow arrow-head displays long and thicker appendages.

Supplementary Figure 5 │ The lactate addition effect on glucose fermentation by C. pasteurianum. The addition of lactate increased butyrate production more than butanol.

References

1.Lee H-S, Krajmalinik-Brown R, Zhang H, Rittmann BE. An electron-flow model can predict complex redox reactions in mixed-culture fermentative BioH2: Microbial ecology evidence. Biotechnol Bioeng 104, 687-697 (2009).

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