Fermenters switch metabolism in microbial fuel cell anodic biofilms

1. Fermenters switch metabolism in microbial fuel cell anodic biofilms Stefano Freguia Bio-analytical and Physical Chemistry Laboratory Kyoto University

2. Background and questions • Can gram-positives express own electrochemical activity? • Previous studies have shown that gram-positive species can utilize exogenous redox compounds for extracellular electron transfer • Lactococcuslactisis a gram-positive homolacticfermenter important in the dairy industry • L. lactisis known to produce several quinone species and exploit them for electron transfer to acceptors such as O2, Fe3+,Cu2+ • However, it lacks a respiratory chain and cannot switch to respiratory metabolism (unless hemes are supplied)

3. Method Potentiostat • Synthetic medium: • - phosphate buffer, pH 7, 100 mM • KCl, 80 mM • NH4Cl, 4 mM • MgSO4·7H2O, 1 mM • Trace elements (nM level) Cathode CE (Pt) Ag/AgCl ref ANODE felt, 100 cm³ Lactococcuslactis added to the medium

4. L. lactisgenerates current from glucose Chronoamperometry E=+100 mV SHE

5. L. lactisexploits 2 diffusive mediators

6. Proposed mechanism of extracellular electron transfer2-amino-3 carboxy-1,4-naphthoquinone (ACNQ) • Soluble quinone due to the carboxy group • E0’=-72 mV SHE e- Anode O OH NH2 NH2 COOH COOH O OH Lactococcus lactis

7. ACNQ versus L. lactismediator

8. L. lactisexploits exogenous ACNQ

9. ACNQ excretion theory is supported by HPLC-UV ACNQ quinol ACNQ quinone 6.88 min spectrum Approximately 200 nM ACNQ quinol in the bulk liquid

10. L. lactismetabolism in the presence of a polarized anode Glucose Max 5% of the substrate electrons are diverted to the electrochemical metabolism in pure culture ACNQox 2NAD+ 2ATP ACNQred 2NADH 2ADP 2NADH 2Pyruvate 42% ACNQox 2NAD+ 2NAD+ ACNQred 2NADH 2Lactate 2Acetyl-CoA 2ADP e- 2ATP 26% 2Acetate 32%

11. Potential implications on a mixed anodic biofilm Glucose Normal fermenters Electrochemically active fermenters H2 e- Acetate Methanogens Acetate CH4 Anodophilic (respiring) bacteria ANODE • Aeration of young biofilms can help to shift towards full conversion of glucose to electricity by: • Killing methanogens • Promoting quinone production in fermenters

12. Conclusions • Gram-positives can self-mediate extracellular electron transfer to anodes • Lactococcuslactisexcretes soluble quinone 2-amino-3-carboxy-1,4-naphthoquinone to mediate electron transfer • L. lactisgenerates redox equivalents by switching from lactate to acetate fermentation • Fermentative bacteria actively contribute to anodic biofilm electrochemical activity • “Clever” operation techniques can lead to methane-free MFCs