Project: Detection and Monitoring of MVOCs

"...Currently, the most frequently cited possibility for the detection of microbial contamination is to use microbially-generated volatile organic compounds (MVOCs) as symptoms of contamination. MVOCs are produced as a metabolic by-product of bacteria and fungi and are detectable before any visible signs of microbial growth appear. MVOCs can therefore serve as early indicators of potential biocontamination problems. Using MVOC analysis, it is also possible to identify specific microorganisms comprising a biomass, determine exposure to and assess potential toxicity from individual MVOC chemicals, and predict the metabolic production of certain mycotoxins.

"Current detection methods rely on cumbersome and expensive gas chromatography/mass spectrometry (GC/MS) and high performance liquid chromatography (HPLC) techniques. As an alternative, we developed a proof-of-concept whole cell bioluminescent bioreporter for the detection and monitoring of the MVOC p-cymene. Bioluminescent bioreporters generate visible light in response to specific chemical or physical agents in their environment. The light response occurs because of the transcriptional activation of a genetically incorporated lux cassette. Because the bioluminescent response is strictly intrinsic to the bioreporter, monitoring is performed autonomously with no user intervention required. The use of these bioreporters will allow rapid and early sentinel detection of biological agents, which will in turn accelerate response time for protective measures.

"Detection of Para-Cymene, a Microbial-Generated Volatile Organic Compound (MVOC), by use of a bioluminescent biosensor. E. Worthington1, K. Daumer2, J. Garland2. 1Dept. of Chemistry Southern Oregon University Ashland OR 97502 and 2Dynamac Corporation Mail Code DYN 3 Kennedy Space Center FL 32899

The purpose of this project was to test procedures and protocols for the development of an integrated biosensor for the detection of para-cymene using bioluminescent bioreporter bacteria suspended in a permeable bead. Research was conducted using two cultures of a strain of Pseudomonas fluorescens developed by the Center for Environmental Biotechnology (CEB) as bioreporters. The preponderance of the project was spent using a bioreporter strain defective for the recombinant gene of interest. The work with the defective strain served to improve contamination control. Work with the second strain provided immediate success and validated the work done with the first strain. A light tight sealed reaction chamber was used to observe the bioluminescence triggered by the metabolism of p-cymene by P. fluorescens Bioluminescence was determined by measuring the current generated by the emitted photons in a photo-multiplier tube. Initial bioluminescence was triggered one hour after the introduction of para-cymene and continued to increase despite a decrease in para-cymene concentration with time. Measurements were obtained at vapor phase concentrations of less than one ppm but a maximum peak emission was not observed at the concentration used Despite the lag in response and lack of correlation between concentration and bioluminescence in this one assay it is still hypothesized that the bioreporter can produce qualitative as well as quantitative detection. Further work will be done to determine the threshold of detection and to develop an accurate flow through detection system. NASA funded this research in conjunction with the Spaceflight and Life Sciences Training Program (SLSTP) at the Kennedy Space Center and in collaboration with the CEB at the University of Tennessee-Knoxville. " - http://asgsb.indstate.edu/programs/2001/34.html