KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association

Proof of principle for a microbial fuel cell biosensor based on Shewanella oneidensis outer membrane protein complexes F. Simonte, F. Golitsch and J. Gescher

Institute of Applied Biosciences, Department of Applied Biology

2 Development of a sensor strain:

1 Abstract:

3.1 Development of a measuring protocol:

3.2 Results:

Fig. 2: Representative U–I curve from S. oneidensis MtrFAB strain. The horizontal line represents the potential of 750 mV vs. NHE.

Fig. 3: a) The current density at the anode was increased continuously and the reached potentials were recorded. Solid lines are representative plots. b) Limiting current density was determined by measuring the current density when the potential reached +750 mV vs. NHE and plotted against the arabinose concentration. A linear dependency from 0.1 mM to 1 mM arabinose is illustrated by a regression curve (y=32.99x+1.98; r2=0.92). Error bars indicate standard deviation from triplicate experiments.

4 Versatility and applications:

These results prove the principle, that S. oneidensis MtrFAB can be applied as a

bioelectrochemical sensor

3 Biosensor performance:

5 References:

www.kit.edu

a)carbon anode

MtrF

carbon anode

Engineering of the strain

Microbial biosensors are advancing biotechnological systems. They offer inexpensive, accurate and easy to operate monitoring possibilities. In this study we present a specific bioanode based sensor that can be adapted to many different analytes. Versatility can be gained by replacing the promoter region of a gene cluster that holds the genetic information for proteins that translate the concentration of an analyte into a current signal. The organism that was genetically engineered to perform this concentration-current conversion is Shewanella oneidensis. This γ-proteobacterium is a well known exoelectrogenic model organism holding an extended respiratory chain to the cell surface.

repX

Px

sugars

organic acids

hormons

promoters sensitive to:

Fig.1: Model of the extended respiratory chain of a) the wild type strain S. oneidensis and b) the engineered strain S. oneidensis MtrFAB. C-type cytochrome proteins are depicted in dark gray. OM = outer membrane, CM = cytoplasmic membrane. A schematic view of the mtr-gene clusters of the respective strains is shown at the bottom of a) and b). Genes coding for c-type cytochromes are depicted in gray. The gene araC codes for the repressor/activator protein AraC that is interacting with the PBAD promoter (symbolized by a black arrow).

The biosensor S. oneidensis strain was constructed by using an outer membrane cytochrome deficient strain. In this background the genes encoding MtrF, MtrA and MtrB were inserted into the genome. They were engineered to be localized adjacent to each other and were set under control of the arabinose inducible promoter PBAD. This resulted in the strain S. oneidensis MtrFAB.

Ferric iron reduction experiments with the biosensor strain S. oneidensis MtrFAB showed a dependency between the inductor concentrations and the rate of ferric iron reduction. Encouraged by these results, a measuring protocol was developed to analyze the possible dependency between inductor level and current production in a bioelectrical setup.

The setup was controlled by a potentiostat to apply a linear increase of current to the system. The bacteria answered with a characteristic U-I curve (Fig. 2).

● The current density at which bacteria fail to provide sufficient electrons to sustain a given current abstraction is characterised by a steep increase in slope of the U-I plot.

● This current at 750 mV was defined as the limiting current density (LCD). The LCD was shown to be characteristic for differing strains and induction levels.

● Current production was correlated to increasing inducer concentrations from 0.1 mM to 3 mM arabinose

● This correlation between induction level and LCD is linear over a wide range of inducer concentrations

a)

a)

b)

b)

1) Bücking et al., Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid-phase terminal electron acceptors. FEMS, 2010.

2) Golitsch et al., Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes. Biosens. Bioelectron., 2013.

Our approach allows to sens several substances, which can enter the cell, just by exchanging the promoter region:

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