Abstract
Monitoring in vitro the metabolic activity of microorganisms aids bioprocesses and enables better understanding of microbial metabolism. Redox mediators can be used for this purpose via different electrochemical techniques that are either complex or only provide non-continuous data. Hydrodynamic chronoamperometry using a rotating disc electrode (RDE) can alleviate these issues but was seldom used and is poorly characterized. The kinetics of Faecalibacterium prausnitzii A2-165, a beneficial gut microbe, were determined using a RDE with riboflavin as redox probe. This butyrate producer anaerobically ferments glucose and reduces riboflavin whose continuous monitoring on a RDE provided highly accurate kinetic measurements of its metabolism, even at low cell densities. The metabolic reaction rate increased linearly over a broad range of cell concentrations (9 × 104 to 5 × 107 cells.mL−1). Apparent Michaelis-Menten kinetics was observed with respect to riboflavin (KM = 6 μM; kcat = 5.3×105 s−1, at 37 °C) and glucose (KM = 6 μM; kcat = 2.4 × 105 s−1). The short temporal resolution allows continuous monitoring of fast cellular events such as kinetics inhibition with butyrate. Furthermore, we detected for the first time riboflavin reduction by another potential probiotic, Butyricicoccus pullicaecorum. The ability of the RDE for fast, accurate, simple and continuous measurements makes it an ad hoc tool for assessing bioprocesses at high resolution.
Highlights
Monitoring in vitro the metabolic activity of microorganisms aids bioprocesses and enables better understanding of microbial metabolism
During microbial oxidation or reduction of a redox probe, a color change can be detected by a spectrophotometer[1,2] and translated into a reaction rate for the pathway under study. (Bio)electrochemical sensors could contribute to microbial kinetic monitoring, with existing examples including toxicity sensing of pesticides, contaminants or antibiotics[3,4,5,6], as well as fast analysis of biochemical oxygen demand of waste water[7,8,9]
The RF/RFH2 couple was electrochemically characterized on glassy carbon (GC) rotating disc electrode (RDE) (Supplementary Discussion S1)
Summary
Monitoring in vitro the metabolic activity of microorganisms aids bioprocesses and enables better understanding of microbial metabolism. (Bio)electrochemical sensors could contribute to microbial kinetic monitoring, with existing examples including toxicity sensing of pesticides, contaminants or antibiotics[3,4,5,6], as well as fast analysis of biochemical oxygen demand of waste water[7,8,9] They have been used in vitro in conjunction with soluble redox mediators to probe the metabolic redox activity of living microorganisms, either immobilized or in suspension[10]. Fresh medium containing substrate(s) and oxidized redox mediator(s) is continuously provided through microchannels in a reaction chamber, where bacterial cells reduce the mediator, which is transduced to an electrical signal on the electrode These elegant devices can efficiently assess the relative impact of some applied stimuli on metabolic reaction rates with very low amounts of microorganisms, allowing low sample consumption[17]. Extracting accurate kinetic information requires a measurement in a reaction free sample, e.g. after centrifugation to remove the biocatalyst[4,5,7,8,12,21]
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