A FRET biosensor for measuring the effectiveness of remediation of heavy metals contaminated soils

The application of biochar as an amendment to contaminated soils can reduce the bioavailability of the metals to microorganisms, by adsorption and other chemical mechanisms. This can potentially decrease heavy metal toxicity, thus improving soil function and productivity. To assess the performance of biochar for the remediation of heavy metal-contaminated soil, a bacterial biosensor was developed in this study to monitor metal bioavailability at the cellular level. In contrast to chemical extraction methods, which measure heavy metal concentrations in operationally defined soil phases, biosensors can produce signals to measure the biologically-relevant impact of heavy metals on cell physiology. A FRET (Förster Resonance Energy Transfer) biosensor was constructed to measure the bioavailability of heavy metals in contaminated soil amended with biochar and compost. This is an attractive approach for the quantification of free heavy metals within living cells. The biosensor consists of a metallothionein protein (which binds Pb2+, Cd2+ and Zn2+), inserted between cyan (eCFP) and yellow (Venus) fluorescent proteins expressed in bacteria cell. In vitro tests of fluorescence emission ratio (VenusFP/eCFP) indicated that the addition of metals to the purified biosensor enhanced FRET between the two fluorescent proteins. The sensitivity of the biosensor was greater for Pb2+ followed by Cd2+ and Zn2+. The FRET biosensor has been expressed in Escherichia coli and Pseudomonas putida, showing its ability to measure heavy metal concentrations in the cytoplasm. The analysis of Ka and FRET ratio maximum parameters revealed that the metal concentrations inside the cells were not the same as those outsides (in metal solution), indicating the measurement of bioavailability. This research has demonstrated the use of a FRET biosensor inside P.putida to measure the bioavailable heavy metals in contaminated soil samples. In situ measurement of the biosensor showed a reduction in heavy metal bioavailability in the biochar-amended soil. The reduction was enhanced in soil amended with a mixture of biochar and compost. The results from the biosensors were supported by a decrease in soluble metals (estimated by chemical extraction) and an increase in soil respiration, suggesting an improvement in soil quality, following the addition of the organic amendments. The research has demonstrated that the FRET biosensor can be used as a complementary tool to assess the remediation performance of biochar more precisely.