An investigation into the use of the yeast Arxula adeninivorans as the catalyst for a microbial fuel cell
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Microbial fuel cells (MFCs) are bioelectrochemical systems which convert chemical energy in organic matter into electrical energy. MFCs use a living microorganism or consortium thereof to metabolise an organic substrate, whereupon some of the electrons the organisms derive from this process are transferred to the anode of the MFC. These electrons will travel hrough an external circuit to a cathode, performing electrical work in the process.
Several yeasts have been investigated for their potential as microbial catalysts because of their robustness when subjected to physical and chemical stresses, coupled with their ability to metabolise a wide variety of organic substrates. One such yeast is Arxula adeninivorans, a dimorphic yeast which has been successfully used as the microbial catalyst of an MFC. It was noticed that A. adeninivorans was able to transfer electrons to the anode of the MFC in the absence of an artificial mediator by means of an unidentified, electrochemically active molecule secreted into the buffer solution.
In this thesis, high-performance liquid chromatography was used to fractionate extracellular buffer from suspensions of A. adeninivorans in order to isolate and collect the unidentified molecule. The molecule was then identified by mass spectrometry as being uric acid. This identification was supported by comparing UV spectra and cyclic voltammograms (CVs) to sodium urate. A transgenic strain of A. adeninivorans with an inducible urate oxidase gene was used to show that the molecule was a substrate for the enzyme urate oxidase, further confirming its identity.
CVs from a range of other yeasts also of the Saccharomycetaceae family were compared to those of A. adeninivorans, and it was observed that none of these other yeasts produced an electrochemical response indicating the presence of uric acid. A transgenic strain of A. adeninivorans with a disrupted xanthine dehydrogenase gene also failed to produce the electrochemical response corresponding to uric acid, indicating that the secreted uric acid is derived from the purine catabolism pathway.
To optimise production of uric acid by A. adeninivorans, linear sweep voltammetry (LSV) was used to measure the concentration of uric acid accumulated over 20 h in the extracellular buffer of A. adeninivorans cell suspensions subjected to a variety of culture conditions. More uric acid was accumulated in suspensions incubated under anaerobic conditions, but less was accumulated by cells derived from poorly-aerated growth cultures compared to well-aerated cultures. The presence of a carbon source in the suspension caused less uric acid to be accumulated. More uric acid was accumulated at 37 °C by suspensions of a mutant A. adeninivorans with a disrupted urate oxidase gene compared to the wild type, but while production increased for both strains at 45 °C (at the cost of greatly increased cell mortality), the wild type accumulated more at this temperature. A pH of 8 was found to be optimal for uric acid production, but the yeast naturally reduces the pH of the buffer over time. In all cases, parameters resulting in increased uric acid production were accompanied by some drawback making it unsuitable for implementation in an MFC.
An MFC was constructed incorporating carbon cloth electrodes, a cation-exchange membrane in place of Nafion, and Dynawhite (3-5% NaOCl) as a chemical catholyte. The anolyte solution was an OD600 2.5 suspension of A. adeninivorans in PBS. A peak power density of 25 mW m-1 with an external load of 3 kΩ was observed. With an external load of 100 Ω, power density diminished rapidly, in contrast to descriptions of such MFCs in the literature. Dynawhite and the cation-exchange membrane functioned acceptably in the short term, but extended use of the two in conjunction resulted in the degradation and failure of the membrane.