Detection of Para-Cymene, a Microbial-Generated Volatile Organic Compound (MVOC), By Use of a Bioluminescent Biosensor, a Proof of Concept

All environments inhabited by humans contain microbes. The detection and elimination of harmful fungi and bacteria in closed system environments such as spacecraft is of vital importance. Biological waste products of microbes can cause “sick building syndrome,” structural damage, and pose a fire hazard. Traditional means of detecting and quantifying these microbes, GC/MS and HPLC, are too bulky and labor intensive to operate in space. Biosensors provide an alternative to conventional instrumentation. This study examined the use of bioluminescent biosensors for the detection and quantification of microbial-generated volatile organic compounds (MVOC’s). A strain of Pseudomonas fluorescens containing the lux operon from Vibrio ficsheri was suspended in an alginate bead and exposed to para-cymene. The MVOC, para-cymene, can be metabolized by P. fluorescens. This strain has been engineered to stimulate activation of the lux operon resulting in the production of bioluminescence with para-cymene metabolism. A non-linear qualitative but perhaps non-quantifiable relationship was observed between para-cymene concentration and electric current generated by bioluminescence. Introduction This work was in support of Dr. Jay Garland’s project “Calibration and Stability Testing of a Microbial Volatile Organic Contaminant (MVOC) Biosensor.” Human habitation is always concomitant with microbial habitation. These microbes, including fungi and bacteria, produce metabolic wastes. On Earth these wastes can be detrimental but the presence of an open system mitigates these effects. In closed systems such as the International Space Station, shuttle orbiters, and any future manned Mars missions, the potential for microbial induced problems is serious. A principal group of metabolic waste products from fungi and some bacteria is volatile organic compounds, (VOC’s).1,2 Many of these MVOC’s, especially terpenes, are unique products of specific fungi or bacteria.1 On Earth these compounds have been found to be responsible for “sick building syndrome” as well as acting as a corrosive agent upon structural materials.2 In space these problems will be more severe. Most organic compounds are combustible, which produces a potential for fires in the high oxygen environments encountered in space vehicles. The traditional means of determining the presence and identity of these compounds has been by use of gas chromatography-mass spectrometry, (GC/MS), or high performance liquid chromatography, (HPLC).1,2,3 Both of these systems, while very accurate, are bulky and high mass, which are limiting factors with current propulsion technology. Additionally, these systems require extensive crew time and do not even provide real time detection or analysis of bio-contaminants. In spacecraft air filtration and surface disinfection will be key in eliminating MVOC’s and microbial growth. Early detection of these compounds is then the critical issue. Chemical weapon sensing is another area in which simply determining presence or absence of a compound is important. Both the American and the British military have developed biosensors that can detect minute quantities of chemical agents.3,4 The British biosensor is based on bioluminescence of a genetically modified bacterium. A similar system could be developed for real time early detection of MVOC biocontamination. Bioluminescence is the process of light emission by a living organism. In bacteria it is produced by the oxidation of reduced flavin mononucleotide, (FMNH2), and a fatty aldehyde in the presence of molecular oxygen catalyzed by the enzyme luciferase.5 Bioluminescence is extremely rare in terrestrial bacteria but more common in marine species. The genes responsible for bioluminescence in one aquatic bacterium, Vibrio ficsheri, have a particularly well-characterized sequence known as the lux gene cassette.4,5,6 This cassette, or operon, consists of five structural genes, luxCDABE, which encode for both luciferase and a multi-enzyme complex that catalyzes aldehyde biosynthesis.2,5,6 The cassette is fully self-contained, meaning no exogenous substrate nor cell lysis is required to achieve bioluminescence.2,6 A – 1 – Edward L. Worthington; Southern Oregon University, Dept of Chemistry, Ashland, OR 97520; ([email protected]) Kathleen Daumer & Dr. Jay Garland; Dynamac Corportation, Life Sciences Support Contract, DYN 3 Kennedy Space Center, FL 32899 recombinant, man-made, version of this gene cassette has been successfully inserted in a number of plasmid cloning vectors with subsequent transformation into and functional expression by several strains of Escheria coli, Psuedomonas aeruginosa and P. fluorescens.6,7 Previous Work The Center for Environmental Biotechnology (CEB) at the University of Tennessee-Knoxville has engineered a bioluminescent strain of P. fluorescens and developed a method by which this bacteria may be incorporated into a biosensor.2,7,8 The P. fluorescens contains plasmid vector pUT mini-Tn5-cym-lux with a cym promoter, the lux gene cassette and genes coding for resistance to both kanamycin and ampicillin. The biosensor was created by suspending the bacteria in a droplet of sodium alginate that was then hardened in a strontium chloride solution.8,9 A metabolic pathway using benzene ring containing compounds is activated in the presence of para-cymene in this bacterium, which triggers bioluminescence.12 Dr. Val Krumins of the NASA Life Sciences Support Contract (LSSC) division of Dynamac Corporation had developed a concept for a flow through analysis system for the biosensor. Drs. D. Kong and L. Levine, also of Dynamac Corporation LSSC division have conducted GC/MS studies of para-cymene at varying concentrations for calibration of the biosensor. Methods and Materials A flow through analysis system was constructed (figure 1). A flow-regulated stream of ultra pure air was bubbled through a solution of para-cymene, ethanol, and distilled water that half filled a 500mL jar. The flow of bubbled air was mixed with and forced the volatilized para-cymene above the solution to flow out of the jar and into the biosensor cell. The biosensor chamber, a light tight jar, contained a fitting for holding the biosensor bead and for holding the tip of the fiber optic light pipe a fixed distance directly above the bead. The biosensor chamber also contained inlet and outlet ports as well as a septum for drawing off headspace gas for GC / MS calibration. Bioluminescence was measured using a fiber optic light pipe (Oriel 77568) coupled with a photo-multiplier tube (Oriel 77340) connected to a multifunctional optical power meter (Oriel 70310) for read out measurements. Downstream of the biosensor chamber was a final jar for measurement of relative humidity and temperature with an outlet to a fume hood. All fittings upstream and within the biosensor chamber were of non-reactive stainless steel or Teflon, while those downstream were a mixture of Teflon, PVC, and surgical tubing. Due to difficulties with rapid loss of para-cymene concentration most measurements were taken in a closed cell while a modified flow through system was engineered. A dark room was constructed using vinyl sheeting and duct tape within the LSSC physiology lab in Hangar L, Cape Canaveral Air Force Station. The P. fluorescens for the biosensor was grown in yeast extract-polypeptone-glucose (YEPG) media treated with kanamycin (Kn), for plasmid selection, overnight at 30oC. The bacteria-YEPG-Kn culture was then be added at a ratio – 2 – Detection of Para-Cymene, a Microbial-Generated Volatile Organic Compound (MVOC), By Use of a Bioluminescent Biosensor, a Proof of Concept