Transcript The Development of Bioluminescent Biosensors for Air Environment
Bioluminescent Sensors for Space Ecosystems
Li Yang
1
, Valentina Kratasyuk
2 1
SLSTP Trainee, Carnegie Mellon University, Pittsburgh, PA, 15213,
2
Professor 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.
Introduction
Bioluminescent Sensors for Space Biotechnology
As NASA embarks on a new era of human space exploration, the 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 monitoring of conditions inside closed ecosystems. The biological 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.
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
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) FMNH2 + RCHO + O2 Luciferase
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.
Methods and Materials for Developing Bioluminescent Detection Systems
Bioluminescent Test Systems
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.
Light Intensity Signal PerkinElmer Bioluminometer
These two bioluminescent tests were assessed on the PerkinElmer Victor2 Bioluminometer along with environmental samples from Kennedy Space Center (KSC).
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 -5 of 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.
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.
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 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.
-5
into a microplate and the
Development of Bioluminescent Test Systems
This experiment establishes standard experimental procedures for conducting bioluminescent tests to monitor contaminants in the water and air of closed environments.
Effect of Common Toxins on Bioluminescent Detection Systems
Fig 1. Effect of Ethanol on E.coli Luminous Bacteria
180000 160000 140000 120000 100000 80000 60000 40000 20000 0 -20000 0 5 10 15 20 25 Luminous Bacteria [10 µl] Luminous Bacteria [20 µl] Luminous Bacteria [40 µl] Luminous Bacteria [60 µl] Luminous Bacteria [80 µl] Luminous Bacteria [100 µl] Luminous Bacteria [120 µl] Luminous Bacteria [140 µl]
Ethanol [µl]
The bacterial system exposed to ethanol showed very pronounced reduction in light intensity (Fig.1).
Fig. 2 Liquid Buthenol on E.coli Luminous Bacteria
700000 600000 500000 400000 300000 200000 100000 0 0 2 4 6
Buthenol [µl]
8 10 12
Fig. 3 Liquid Butenol on the Coupled Enzyme System
7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0 1 2 3
Buthenol [µl]
4 5 6 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.
Obtaining the Coupled Enzyme Reaction Mixture
Fig 5. Influence of Ethanol on FM N Fig 4. Influence of Ethanol on Aldehyde
1400000 1400000 1200000 1000000 800000 600000 400000 200000 0 0.
5 2.
5 4.
5 6.
5 8.
5 10 .5
12 .5
14 .5
16 .5
18 .5
Aldehyde [µl]
20 .5
22 .5
No Ethanol Ethanol 1 µl 1200000 1000000 800000 600000 400000 200000 0 0 5 10 15
FMN [µl]
20 25 30 No Ethanol Ethanol 1 µl 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.
Results and Discussion
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..
Bioluminescent Assessments of Environmental Samples Bioluminescent Assessment of Gasses
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.
Water Filtration Assessment
10000000 1000000 100000 10000 1000 100 10 1
Fig 6. Bioluminescent Assay of the Capacity of Filters to Eliminate Bacteria
Bacteria Filtered Bacteria Broth
Fig. 10 Bioluminescent Assay of Buthenol Gas on E.coli Bacteria System [Injection into Vial ]
1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 0 50 100
Gas [µl]
150 200 Air 100 µl Buthenol Gas
Fig 11. Bioluminescent Assay of Buthenol Gas on E.coli Bacteria System [Direct Injection ]
2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 0 2
Gas [µl]
4 6 Air Buthenol 0 10^-6 10^-5 10^-4 10^-3 10^-2
Bacteria Concentration
10^-1 1 Butanol gas was found to produce a slight inhibition of the enzymatic reaction (Fig. 12).
Bioluminescent assessment of NanoCeram filters showed that the filters were capable of filtering bacteria (Fig. 6).
Fig 12. Bioluminescent Assay of Butenol Gas on Coupled Enzyme System
Nutrient Water Bioluminescent Assessment
Fig. 7 Bioluminescent Assessement of Growth Chamber Water Filtration
10000000 1000000 100000 10000 1000 100 10 1 Water Filtered Water Bacteria 0 10^-6 10^-5 10^-4 10^-3 10^-2 10^-1 1
Bacteria Concentration
100000000 10000000 1000000 100000 10000 1000 100 10 1 0 200 400 600 800
Time [s]
1000 1200 1400 1600
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.
Fig 9. Luminescence of Concentrations of E.coli Luminous Bacteria Fig 8. Optical Density of Concentrations of E.coli Luminous Bacteria
10000000 1000000 100000 10000 1000 100 10 1 1.2
1 0.8
0.6
0.4
0.2
0 0 10^ 6 10^ 5 10^ 4 10^ 3 10^ 2
Bacteria Concentration
10^ 1 1 0 10^-6 10^-5 10^-4 10^-3 10^-2 10^-1
Bacteria Concentration
1 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.
A Nutrient Plant Soil Seed Medium was investigated by a luminous bacteria assay (Fig. 13, 14).
Fig 13. Bioluminescent Assay of the Nutrient Seed Medium (linear)
2500000 2000000 1500000 1000000 500000 0 0 0.1
0.2
0.3
0.4
Mass [g]
0.5
0.6
0.7
0.8
Fig. 14 Bioluminescent Assay of the Nutrient Seed Medium (logarithmic )
10000000 1000000 100000 10000 1000 100 10 1 0 0.1
0.2
0.3
0.4
Mass [g]
0.5
0.6
0.7
0.8
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.
Conclusions
• 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.
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 biotechnology”.
Biosensors for Space
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.
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.
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.