Li Yang 1 , Valentina Kratasyuk 2

1. Bioluminescent Sensors for Space Ecosystems Li Yang1, Valentina Kratasyuk2 1SLSTP Trainee, Carnegie Mellon University, Pittsburgh, PA, 15213, 2Professor of Biophysics, Krasnoyarsk State University, Krasnoyarsk, Russia Abstract Bioluminescent assays were conducted to monitor the toxicity of contaminants in air, water, and soil samples taken from environmental chambers located at the Space Life Science Laboratory (SLSL) at Kennedy Space Center. Two methods were developed to monitor contaminants in closed ecological systems. They consisted of an in vivo assay using luminous bacteria, and an in vitro assay using the coupled enzyme system NADH:FMN-oxidoreductase-luciferase. The bioluminescent assays were used to detect the contaminants in samples of water and gas. The luciferase enzyme system was found to have more sensitivity to ethanol than the bacteria system. Bioluminescent methods for the control of liquid filters were developed with luminous bacteria. • Bioluminescent methods were developed in this research to monitor the air, water, and soil samples in closed ecosystems. These methods serve as a set of guidelines for conducting bioluminescent toxicity tests for closed environments. • Bioluminescent tests were found to be capable of detecting toxins in the liquid solutions. • The bioluminescent systems were found to have high sensitivity to minute amounts of liquid ethanol (1µl) and butanol (2ul), which are common cleaners on space shuttles. • New bioluminescent methods for control of the purification of water were developed with NanoCeram filters. • The coupled enzyme system was slightly inhibited by an air sample of 500 ppm of buthanol gas. Conclusions Methods and Materials for Developing Bioluminescent Detection Systems Bioluminescent Test Systems KSC Growth Chambers Liquid:Bioluminescent Assessment of a Water Filtration System The bioluminescent test system consisted of 100 µl E.coli bacteria in control in dilutions from 1 to 10-5of Bioluminescent Bacteria System To find the volume for maximum light intensity, E.coli bacteria dissolved in tryptic soy broth was pipeted in volumes from 10 to 140 µl into a microplate. The effect of different mediums [water and ethanol] was tested with different volumes of liquids from 0 to 100 µl. Coupled Enzyme Test System The enzymatic reaction mixture contained 10 µl 0.002% Aldehyde solution, 5 ul 0.04 mM FMN, 2-5 µl Luciferase-oxidoreductase (1 ml phosphate buffer added to vial of lyophilized enzymes), 20 µl phostphate buffer pH 6.9, 10 µl NADH. To determine effect of common toxins on the coupled ezyme system, buthanol was injected to the reaction mixture in concentrations from 0 to 10 µl. To determine the reaction mixture, 1 µl ethanol was injected into enzymatic reaction mixture with varying amounts of FMN and Aldehyde. the bacterial solution. 80 ml of 1/3 Hoaglands water was collected from KSC growth chambers. Nutrient water was injected into the bioluminescent bacterial solutions on the microplate. Light intensity readings were taken with the bioluminometer to determine the steady state curve. A 5ml syringe with NanoCeram filters from Argonide Co. was used to filter the growth chamber water. Filtered water was pipeted on the microplate to see if bioluminescent test system can assess the filtration of water. Bacterial solution was filtered with NanoCeram and pipeted onto the microplate. The number of bacteria was calcula- ted from their optical density at a 600 nm setting using a Genesis 20 spectrophotometer to be compared with light intensity readings from the bioluminometer. Introduction Gass: Bioluminescent Assessment of Toxic Gasses Gas samples of 1000 ppm ethanol and 500 ppm buthanol were harnessed from the VOC project at the SLS lab. E.coli with LUX-gene dissolved in tryptic soy broth solution was prepared in concentrations from 1 to 10-5. Gas samples were directly injected into the microplate and indirectly injected in a sealed 2 ml vial. Light intensity I (t) was measured after definite time intervals for the duration of 1-2 min. The changes in light intensity I(0)/I(t)*100 % were correlated with toxicity of the air samples to biological organisms. Future Work These investigations of bioluminescent assays indicate the advantages of using bioluminescence in applications for space biotechnology. This result will be used to develop a proposal entitled “Bioluminescent Biosensors for Space biotechnology”. The future prospects of this research is directed to the development of bioluminescent systems to control levels of contaminants in the air, water, and soil of closed ecological life support systems. In addition, bioluminescent methods for control of water filtration will be developed in collaboration with the Argonide Corporation. Bioluminescent Sensors for Space Biotechnology environmental control of closed ecosystems will be crucial for the long-term success of missions into space since they maintain essential life support functions to sustain a human crew during space flight. It will be of crucial importance to develop biological sensors to monitor the environmental conditions inside closed ecosystems. The biological As NASA embarks on a new era of human space exploration, the These two bioluminescent tests were assessed on the PerkinElmer Victor2 Bioluminometer along with environmental samples from Kennedy Space Center (KSC). Light Intensity Signal PerkinElmer Bioluminometer Soil Seed Medium: Bioluminescent Test of Seed Medium for Soil The seed medium fiber was cut into pieces, massed from 0.1 to 0.7 g, and soaked in 10 ml of dionized water overnight. 50 µl of E.coli bacteria was pipeted in concentrations from 1 to 10-5 into a microplate and the initial light intensity was measured. 20 µl seed medium solution was injected into the bacteria after 1-2 min and the new light intensity was recorded. monitoring of the environmental constituents of closed ecological systems can be accomplished by bioluminescent detection. In the past, bioluminescent sensors have been developed to monitor natural aquatic ecosystems [1,2,4,5,6]. These methods were adapted to monitor systems used for human space travel. Results and Discussion Bioluminescent Assessment of Gasses Development of Bioluminescent Test Systems References 1. Farre M., Barcelo D. Toxicity Testing of waste water and sewage sludge by Biosensors, bioassays and chemical analysis. Trends in Analytical Chemistry, Vol. 22, No. 5, 2003. 2. Kratasyuk V.A. Esimbekova E.N. Polymeric Biomaterials, The PBM Series, V.1:Introduction to Polymeric Biomaterials, Arshady R Ed, Citus Books, London, pp. 301-343, 2003. 3. Kratasyuk V.A., et al. The use of bioluminescent bio-tests for study of Natural and laboratory aquatic ecosystems. Chemosphere, 42: 909-915, 2001. 4. Kratasyuk V. A., et al. Bioluminescent water quality monitoring of salt lake Shira. Luminescence; 14: 193-195, 1999. 5. Paddle, Brian. Biosensors for Chemical and Biological agents of defense Interest. Review Article. Biosensors and Bioelectronics Vol. 11 No. 11:1079-113, 1996. 6. Vetrova E., Bioluminescence characteristics of Lake Shira water. Aquatic Ecology 36: 309-315, 2002. Gas samples (5000 ppm Butanol) were injected to the bioluminescent systems by two methods: (1) the gases were bubbled to 2 ml luminous bacteria solution (Fig. 10); (2) the gases were directly injected into the microplate (Fig. 11). Butanol was found to have a slightly excitatory effect on the bacteria. The standard reaction mixture of coupled enzyme system was determined: 10 µl 0.002% tetradecanal, 5 µl 0.04 mM FMN, 2 µl Luciferase-Oxidoreductase, 20 µl Phosphate buffer, 10 µl NADH.. This experiment establishes standard experimental procedures for conducting bioluminescent tests to monitor contaminants in the water and air of closed environments. Objectives • This project aims to develop sensitive, low cost, versatile bioluminescent sensors capable of monitoring multiple aspects of the internal environment in closed ecological space life support systems. The objectives for this study were six fold: • to develop the biological component of bioluminescent sensors to monitor closed environment of space ecosystems. • to find the conditions (the amount of luminous bacteria and concentrations of enzymes, flavin mononucleotide FMN, tetradecanal aldehyde, NADH) to conduct environmental toxicity assays • to investigate the sensitivity of these bioluminescent test systems on model pollutants (ethanol and buthanol) • to investigate the nutrient water from environmental growth chambers and the process of its purification with NanoCeram filters • to develop the methods of gas pollutant detection • to develop assays for control of water filtration systems in environmental growth chambers Bioluminescent Assessments of Environmental Samples Effect of Common Toxins on Bioluminescent Detection Systems Water Filtration Assessment Butanol gas was found to produce a slight inhibition of the enzymatic reaction (Fig. 12). The bacterial system exposed to ethanol showed very pronounced reduction in light intensity (Fig.1). Bioluminescent assessment of NanoCeram filters showed that the filters were capable of filtering bacteria (Fig. 6). Nutrient Water Bioluminescent Assessment Acknowledgements This research was conducted as a part of the 2005 Spaceflight and Life Sciences Training Program funded by the National Aeronautics and Space Administration. The authors recognize the support of the Dynamac Corporation, the NASA Spaceflight and Life Sciences Training Program Academic Partner Alliance and the United States Department of Agriculture. Thanks to Diane Shoeman, SIFT (Summer Industrial Fellowships for Teachers) and Frank Mycroft (SLSTP trainee) for conducting parallel laboratory research, Dr. Ignascio Eraso for providing samples of buthanol and ethanol, Dr. Micheal Roberts and Michelle Birmele for assistance with the PerkinElmer Victor 2 Bioluminometer, Lashelle E. McCoy for providing samples of 1/3 hoaglands solution from environmental growth chambers. In addition, we would like to thank SLSTP trainees, Antrelle Kid, Jake Elmer, Jonathan for providing laboratory materials essential for performing these experiments. The sensitivity of luminous bacteria and the enzyme system to ethanol (1 µl) was more than for butanol (2 µl) (Fig. 2,3). Therefore, 1µl of ethanol would be used for further experiments. Soil Seed Medium Bioluminescent Assessment The bioluminescence tests found that 1/3 hoaglands nutrient water collected from the environmental chambers at KSC had no toxicity effects on the biological test system (Fig. 7); filtered and unfiltered water showed the same light intensity emissions. A Nutrient Plant Soil Seed Medium was investigated by a luminous bacteria assay (Fig. 13, 14). Methods This research developed two bioluminescent test systems for toxicity assays: the whole cell bacteria and the coupled enzyme system. Coupled Enzymatic Reaction: NADH:FMN-oxidoreductase NADH (NADPH) + H+ + FMN  NAD(NADP)+ + FMNH2 (1) Luciferase FMNH2 + RCHO + O2  FMN + RCOOH + H2О + h (2) ’ The biological systems consisted of bioluminescent bacteria and their enzymatic extracts. Both test systems are based on the coupled enzymatic reaction shown above. Obtaining the Coupled Enzyme Reaction Mixture Light emission intensity was dependent on tetradecanal aldehyde and FMN concentrations (Fig. 4, 5). Ethanol was found to compete with tetradecanal as it reduced the intensity of light emissions with increasing concentrations of aldehyde. This suggests that ethanol acts to disrupt tetradecanal activity in the enzymatic reaction. Ethanol did not influence the FMN substrate activity. The nutrient soil solution has no toxic effect on the bioluminescent system. This proves to be good for using the bioluminescent system for toxicity testing. These results along with previous results also show that while our biosensor is sensitive to toxins like ethanol and butanol it is not affected by environmental materials like nutrient water or soil seed mediums. To determine if luminous bacteria could be used as an accurate measure of water filtration, the luminescence of the unfiltered bacteria (Fig. 9) was compared to their optical density (Fig. 8). The comparison showed that luminous bacteria test was highly sensitive.