DEVELOPMENT AND CHARACTERIZATION OF A WHOLE-CELL BIOLUMINESCENT SENSOR FOR BIOAVAILABLE MIDDLE-CHAIN ALKANES IN CONTAMINATED GROUNDWATER SAMPLES

A microbial whole-cell biosensor was developed, and its potential to m easure water-dissolved concentrations of middle-chain-length alkanes a nd some related compounds by bioluminescence was characterized, The bi osensor strain Escherichia coli DH5 alpha(pGEc74, pJAMA7) carried the regulatory gene alkS from Pseudomonas oleovorans and a transcriptional fusion of P-alkB from the same strain with the promoterless luciferas e luxAB genes from Vibrio harveyi on two separately introduced plasmid s, In standardized assays, the biosensor cells were readily inducible with octane, a typical inducer of the alk system, Light emission after induction periods of more than 15 min correlated well with octane con centration, In well-defined aqueous samples, there was a linear relati onship between light output and octane concentrations between 24 and 1 00 nM, The biosensor responded to middle-chain-length alkanes but not to alicyclic or aromatic compounds. In order to test its applicability for analyzing environmentally relevant samples, the biosensor was use d to detect the bioavailable concentration of alkanes in heating oil-c ontaminated groundwater samples, By the extrapolation of calibrated li ght output data to low octane concentrations with a hyperbolic functio n, a total inducer concentration of about 3 nM in octane equivalents w as estimated. The whole-cell biosensor tended to underestimate the alk ane concentration in the groundwater samples by about 25%, possibly be cause of the presence of unknown inhibitors, This was corrected for by spiking the samples,vith a known amount of an octane standard. Biosen sor measurements of alkane concentrations were further verified by com paring them with the results of chemical analyses.