Characterisation of novel electroactive consortia from environmental samples enriched through potentiostatic growth
Doyle, Lucinda Elizabeth
Date of Issue2017
Interdisciplinary Graduate School (IGS)
Singapore Centre for Environmental Life Sciences Engineering (SCELSE)
Despite the great microbial diversity nature has to offer, a relatively small group of electrochemically-active microorganisms (EAM) has been discovered. This work aimed to explore the use of a three-electrode set-up to enrich and isolate novel EAM. The approach was chosen as an alternative to the conventionally used microbial fuel cell. A three-electrode set-up using a carbon felt working electrode was developed and optimised with a pure culture of Pseudomonas aeruginosa, a species not conventionally tested using this type of system. The platform was sensitive enough to detect current generation using phenazines as redox mediator. Furthermore, simultaneous, real-time electrochemical detection of the redox-active metabolites Pseudomonas quinolone signal and pyocyanin was achieved in live cultures using voltammetry, with implications for diagnostic devices in healthcare settings. After this validation, the platform was then used to characterise a complex microbial community, during long-term electrochemical enrichment. Tropical climate sediment was chosen as inoculum source, as it is currently under-explored in this context which frequently focuses on enrichment from waste water. Specifically, sediments from a canal system and a mangrove region of Singapore were chosen and enriched separately for EAM. Comparison revealed the enriched community from canal sediment was superior to that from the mangrove sediment in terms of electrogenic activity when evaluated by chronoamperometry, and was chosen for further characterisation. Voltamperometric techniques and electrochemical impedance spectroscopy, performed over a wide range of potentials, dynamically characterised extracellular electron transfer (EET) over the four month enrichment. Metagenomics and metatranscriptomics analysis revealed that Geobacter metallireducens was heavily enriched on the electrodes, a species not typically associated with a lactate-fed system such as this. Chlorobium, Clostridium and Rhodopseudomonas were also abundant, indicating tropical climate sediments are host to a range of microorganisms capable of EET. Two novel isolates were grown in pure culture from the enrichment, identified to be from the genera Enterobacter and Aeromonas. Both isolates were capable of EET in defined media on carbon felt and screen-printed electrodes. Both could avail of acetate and lactate as electron donor. Additionally, the Enterobacter isolate could perform mediated electron transfer using the soluble redox shuttle 2- hydroxy-1,4-naphthoquinone (HNQ), indicating it may have availed of this mode of EET during the initial enrichment. This work outlines a comprehensive methodology for characterising and isolating novel EAM from unconventional inocula. It also highlights the applicability of this set-up for monitoring a wide range of EAM, in both mixed communities and pure culture. Specific genera characterised range from high current generators such as Geobacter to more modest performers such as Pseudomonas, Enterobacter and Aeromonas. In addition to characterising EAM, such technology can be used for biosensors, as demonstrated by the detection of redox-active metabolites of the pathogen Pseudomonas aeruginosa.