Clotho Science Program
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Contents |
Background
Introduction
The upper extent and characteristics of the biosphere (the collection of all living things and their interactions) is of keen interest to astrobiologists. The upper atmosphere is an extreme environment that is dry, cold, and exposed to much greater amounts of UV radiation than the earth's surface. The understanding of ability of life to survive in extreme environments such as this is considered critical to understanding and predicting the possibility of life in extraterrestrial environments. Cloudborne microbes are similarly important, potentially even more so since clouds may provide an potentially advantageous aqueous environment.
However, the study of airborne microbes (bioaerosols) began in the fields of human health and occupational hazards, looking at allergens and airborne infections and irritants, and to date almost all existing technology in the field has been developed for use in indoor (human-made) environments such as factories and hospitals. Commercial devices generally use suction to pull a constant flow of air through a sampling chamber which then uses direct impingement, or more indirect methods such as varied settling times (gravity) or average charge distribution (electrostatics) to differentiate by size. Since they are usually intended for use in monitoring a particular, expected aerosol (such as pollen in a greenhouse), they often do not record more than simple particle count and may have very limited size range for accuracy as well; further analysis, such as testing to determine how many of the particles are biological in origin, often requires removal of the sample to a lab for culturing. Typically, these commercial devices are no suitable for outdoor, experimental sampling use; the most significant problem is that almost all are designed to produce reliable results only in isokinetic flow conditions, which typically do not exist in natural environments. They may also lack robustness, capture too narrow of a size range for broad experimentation, or require anchoring to large structures which are impractical or impossible to erect on-site.
Previous Work
As a result, what previous work has been done in bioaerosol capture has focused entirely on capture and return. For example, Imshenetsky et al. [1] used long ribbons of film coated with nutrient carried by a meteorological rocket as high as 77 km, and watched the returned film kept in a warm chamber with a microscope to detect growing colonies of fungi. Wainwright et al. [2] used a balloon-flown cryosampler (chambers cooled with liquid helium) to trap air samples at altitudes up to 41 km and attempted to culture bacteria from the samples. Amato et al. [3] used a large-particle sampler set at the peak of a mountain to capture cloud droplets from which bacteria, yeast and fungi were grown; although clouds provide the most interesting and potentially habitable high-altitude environment, there has been little other work done from a biological standpoint on cloud droplet capture.
Although these results, if accurate, give a general indication that there is microbial life in the upper atmosphere, none give a general picture of the vertical profile of the biosphere: the different types (even broadly speaking) of microbes and their relative densities (even if only at a given instance in time) over the altitude range of the sampling. Additionally, bioaerosols even at ground level are known to have significant seasonal and even diurnal variations, but none of these experiments have been repeated systematically over time to probe these effects.
The previous results also face various questions about their reliability. Capture and return introduces many possible avenues of contamination: the sample, though theoretically isolated, undergoes a descent, long-distance, transport, and multiple transfers and divisions in a lab by multiple technicians. Because all analysis takes place at once after the fact, there is always the possibility that organisms were carried up with the sampler in the first place despite all efforts at sterilization. Capture and return also generally entails study by culturing, which introduces inherent biases in the distribution of results as no known culturing method is (or could be) equally hospitable to all possible forms of microbial life. As Wainwright cautioned when reporting his results, "The only certain means of proving the existence of microorganisms in the stratospheres is to send probes where samples can be analysed in situ."
Technology and Analysis
There is little existing technology specialized for in-situ analysis beyond the standard flow cytometers (particle count), some of which can be combined with stains (or fluorescence) to look particularly for biological specimens. For instance, the staining procedure could be automated and combined with the general size-sorting mechanism of a multi-stage impinger to get a more reliable breakdown of bioaerosols by size. These results could also be compared with later culture and other results to estimate what percentage of captured bioaerols were not culturable using the chosen methods. For longer-term analysis, the samples could be deposited directly onto plates which were photographed using digital microscopy, recording any in situ growth. Other, more difficult possibilities include combining the sampler with something like the recently developed miniaturized PCR machines. Further guidance to later analysis could also be given by taking temperature, humidity, UV, and other data along with the sample. As any of these techniques could be directly combined with subsequent return analysis, they offer only additional advantages.
Goals
This project has two goals in mind. The more straightforward is to determine is the highest altitude at which living material can reliably be detected. The more long-term is to develop a vertical profile of the biosphere, from the earth's surface to the atmosphere's furthest extent. At a minimum, this requires a system capable of capturing a wide size range of particles and able to guarantee that the capture is clean (not contaminated), of sufficient quantity to detect reasonable population thresholds, and viable (if it was viable to begin with). If this is successful, it is immediately extensible to addressing the representativeness of the sample (absolute rather than relative concentration), spatial and temporal variations in population density, and wet (cloud-droplet) as well as dry atmospheric environments. The ultimate goal of the payload design is thus to develop a robust, reliable and reusable system, suitable for use with both balloon and rocket-powered flight, capable of capturing and preserving a wide range of bioaerosols at various altitudes, with appropriate in situ analysis capabilities.
To this end, we have set the following goals for Dec 31, 2010:
- Develop the strategy for sampling sites and times for flights.
- Design, obtain and build environmental instruments and air sampling devices.
- Test science payload package. Testing will take in a series of steps and iterations: lab testing, Airship Ventures, environmental chamber at Ames Research Center, Pacific Northwest National Labs simulation facility with Daniel Cziczo, and (optionally) high altitude balloon.
- Flight testing of science payload on rockets.
- Obtain initial (~3) vertical profiles of environmental data and particle capture.
- Trouble-shoot single cell pcr from captured particulates.
- Begin design of on-board biological processing.
Differences from Previous Studies
- Sampling strategy
- Higher maximum altitude than previous studies
- Long-term commitment / potential for repeated sampling
- Temporal and Vertical profiling
- Correlation of biodiversity with environmental profiling
- Sampling capabilities
- Targeting areas of known biological interest
- State-of-the art air sampler
- Single cell pcr coupled with environmental microscopy
- On-board biological processing
Flight Plans
- Main article: Payload Design
Given the short time schedule and the importance of returning at least some living specimens on the initial flight, we have decided to do whole-air sampling. This is implemented using evacuated or cooled pressure cylinders opened in sequence at specific altitudes. It is a relatively simple and robust technique. For an example, see Indian Journal of Radio & Space Physics 1996 Lal.pdf.
Team and Advisors
Science Payload Team
Dr. Lynn Rothschild, Diana Gentry, Yi Liu
Advisors
References
[1] Imshenetsky et al. "Upper boundary of the biosphere." Appl Environ Microbiol (1978) vol. 35 (1) pp. 1-5, available here: Appl Environ Microbiol 1978 Imshenetsky.pdf
[2] Wainwright et al. "Microorganisms cultured from stratospheric air samples obtained at 41 km." FEMS Microbiology Letters (2003) (218) pp. 161-165, available here: FEMS Microbiology Letters 2003 Wainwright.pdf
[3] Amato et al. "Microbial population in cloud water at the Puy de Dôme: Implications for the chemistry of clouds." Atmospheric Environment (2005) (39) pp. 4143-4153
