Extracellular Electron Transfer of Lactococcus lactis: Fundamentals and Applications

The Gram-positive bacterium, Lactococcus lactis, plays an immensely important industrial role in food fermentation processes. L. lactis normally relies on a fermentative metabolism under anaerobic conditions, where NADH generated by glycolysis is re-oxidized into NAD⁺ by the lactate dehydrogenase, converting more than 90% of the metabolized sugar into lactate. Despite of its fermentative metabolism, L. lactis can also use oxygen as an electron acceptor. There are two ways, in which oxygen can serve as an electron acceptor to oxidize NADH: through the H₂O-forming NADH oxidase (NoxE), or via respiration in the presence of heme, its precursor protoporphyrin IX or hemin. When L. lactis grows with respiration, less ATP is spent on generating the essential proton electrochemical gradient, which increases the biomass yield. For this reason, starter culture manufacturers often harness respiration when culturing L. lactis. However, the use of animal blood, as a source of heme, in microbial food cultures can be unwanted for some customers. Moreover, there are challenges associated with using oxygen as electron acceptor, e.g. its low solubility in the fermentation broth can be a major limitation for microbial growth and production, microorganisms can be oxidatively stressed by it, and supplying oxygen/air is costly as pumping and stirring oxygen into the fermentation broth requires a lot of electrical energy.

The aim of this PhD project was to investigate the possibility of replacing oxygen with an alternative electron acceptor for regeneration of NAD⁺, which has been termed extracellular electron transfer (EET). First, we explored the effects of extracellular electron acceptor ferricyanide on the wild-type L. lactis MG1363 and a mutant of L. lactis, CS4363, blocked in NAD⁺ regeneration. We tested the electron acceptor ferricyanide, which supported good growth of CS4363, which has not been reported previously. When growth was facilitated by EET, we observed that cell morphology was altered from the normal coccoid to a more rod-shaped appearance, and acid resistance was increased. By electrochemical analysis and knock-out of possible relevant genes in EET, we found that the NADH dehydrogenase and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) were important in EET. To enhance the capacity of EET, adaptive laboratory evolution (ALE) was performed for CS4363, and EET-enhanced mutant CS4363-F2 was further characterized physiologically.

To figure out the underlying mechanism of the enhanced capacity for EET in mutant CS4363-F2, the strain was whole-genome sequenced and a comparative transcriptomics analysis was carried out. It was found that the amino acid metabolism and nucleotide metabolism had changed significantly in CS4363-F2. The gene noxB, encoding NADH dehydrogenase, was found to be most upregulated (log₂FC=3.56), and the reason for this was a single-nucleotide variation (SNV) in its promoter region. NoxB was found to differ from the other NADH dehydrogenase, NoxA, of L. lactis, and was demonstrated to be a novel type II NADH dehydrogenase solely involved in EET. A bioinformatics study revealed that NoxB-type NADH dehydrogenases could be found widely distributed in other L. lactis strains and members of the gut microbiota.

Finally, we explored whether it was possible to support the growth of CS4363 and its EET-enhanced mutant by an anode in a bioelectrochemical system (BES) setup. The ALE mutant CS4363-F2 displayed a remarkable performance, producing a high-yield bulk chemical 2,3-butanediol and achieving a record high current density of 0.809±0.051 mA/cm².

This study has shed light on the fundamental mechanism of EET in L. lactis, and has revealed the feasibility of using anodic electro-fermentation (AEF) to grow L. lactis, thereby demonstrating the enormous potential of AEF in the fermentation industry.