A multi-channel continuous toxicity monitoring system using recombinant bioluminescent bacteria for classification of toxicity.

A multi-channel system for continuous toxicity monitoring and classification of toxicity was developed based upon a previously developed two-stage minibioreactor system. The multi-channel system consists of a series of a two-stage minibioreactor systems connected by a fiber optic probe to a luminometer. Each channel was used for cultivating different recombinant bacterial strains, such as TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2540 (fabA::luxCDABE), which are induced by protein-, DNA-, and cell membrane damaging-agents, respectively. GC2 (lac::luxCDABE) is a bacterium expressing bioluminescence constitutively, which shows a reduction in its light level as cellular toxicity increases. Artificial wastewater samples were made by combining toxic chemicals, including Mitomycin C (a representative DNA damaging agent), phenol (a representative protein damaging agent), and cerulenin (a representative cell membrane damaging agent), and injecting this sample into each channel in order to simulate the detection of toxicity for mixed chemical samples. Each channel showed a specific bioluminescent response due to the toxic chemicals contained in the sample wastewater, while GC2 showed a general response to cellular toxicity. By using this multi-channel continuous toxicity monitoring system, classification of toxicity in field samples was found to be possible.

A biosensor for the detection of gas toxicity using a recombinant bioluminescent bacterium.

A whole-cell biosensor was developed for the detection of gas toxicity using a recombinant bioluminescent Escherichia coli harboring a lac::luxCDABE fusion. Immobilization of the cells within LB agar has been done to maintain the activity of the microorganisms and to detect the toxicity of chemicals through the direct contact with gas. Benzene, known as a representative volatile organic compound, was chosen as a sample toxic gas to evaluate the performance of this biosensor based on the bioluminescent response. This biosensor showed a dose-dependent response, and was found to be reproducible. The immobilizing matrices of this biosensor were stored at 4 degrees C and were maintained for at least a month without any noticeable change in its activity. The optimal temperature for sensing was 37 degrees C. A small size of this sensor kit has been successfully fabricated, and found to be applicable as a disposable and portable biosensor to monitor the atmospheric environment of a workplace in which high concentrations of toxic gases could be discharged.

A two-stage minibioreactor system for continuous toxicity monitoring.

A two-stage minibioreactor system was successfully developed for continuous toxicity monitoring. This system consists of two minibioreactors in series. Recombinant Escherichia coli DPD2794 containing a RecA::luxCDABE fusion as a model strain was utilized to monitor environmental insults to DNA, with mitomycin C as a model toxicant. Pulse type exposures were used to evaluate the system's reproducibility and reliability. Step inputs of mitomycin C have been adopted to show the system's stability. The system's ability to monitor the possible upsets or accidental discharges of toxic chemicals was also evaluated with these step insults. All the data demonstrated that this two-stage minibioreactor system using recombinant bacteria containing stress promoters fused with lux genes is quite appropriate for continuous toxicity monitoring. Long-term operation and minimized media-usage have been investigated. Thus application to many different areas, including an early warning system of wastewater biotreatment plant upsets and the monitoring and tracking of accidental spills, discharges or failures in plant operation are plausible.