Update: December 19, 2012 Andrew identified University of Colorado Alpine Microbial Observatory that can do sequencing with support from Craig. Ready to do first test with controls on the making the sterile cotton candy sponge mechanism for collecting DNA out of the air, and need to do so before mid-February. We learned that if we scale up in the future, we can make our own sterile sucrose. Update: November 10, 2012 During EcoHack3, a large group of people including Andrew Hill, Mariko Kosaka, Valerie Farber, JD Godchaux, Alyssa Wright, Craig Mills, David Danziger, Erin ???, Liz Barry, and more worked together to sketch out and early prototype this project. See the Research Note of our blackboard brainstorming. DESCRIPTION OF PROJECT EcoHack III - Living Sky This November, as part of the EcoHack conference, we will work with a portion of the participants to build a kit to sample microbes in the environment in a way never done before, through citizen science and balloon-based sampling. Over the course of Saturday, teams will hack on numerous projects, among those we intend to support a group that will design and test hardware that will enable microbial sampling from helium balloons. The hardware will include apparatus for microbe sampling as well as the collection of environmental data. On a day following Ecohack (to target overcast skies), teams will spread throughout the New York City metro area with balloon sampling kits. Teams will deploy helium filled balloons carrying environmental sensors and microbial sampling kits. Sampling will be done at or around 500 feet. Samples will be retrieved and prepared for transport back to storage and preparation facilities. All samples will be given a unique identifier that will allow them to be linked back to sampling and environmental data collected. Over the course of the following weeks, samples will be sequenced and published online. IMPACTS Airborn microbial populations may have profound impact on the world around us. Some known areas of interest include, climate and weather, human health, agricultural health, and global environmental change. It has been found previously that clouds offer a unique environment for fairly abundant microbial populations (see Amato 2012) and that there are enough microbes in the clouds to affect physicochemical processes. SEQUENCING WORKFLOW coming... Collection Filtering DNA Extraction and Amplification being done by Krista McGuire and lab techs. Extraction is important because when you take an air sample, you collect much more than just DNA. Amplification takes those extracted DNA strands and replicates them. This step tests for presence of particular organisms, not for abundance or concentration. Once amplified, we'll send a small part of that sample off to get sequenced, and freeze the rest. Sequencing and Assembly MICROBE COLLECTION The challenge is designing a balloon mounted device that can collect microbes from the air column. Commonly, air sampling of microbes relies on industrial vacuums with filters. These devices are too large and too expensive for our study (for now). Some other options exist. - Impaction of suspended water droplets on a surface. Drawback is that we would have to perform under cloudy/foggy condition and our collection would not be limited to microbes only. See Mohen, 1989 - Novel filters. The industrial vacuum is needed because traditional filters are very hard to pass air through. Perhaps we can conceive a filter that is 'sticky' enough to collect microbes without being so fine as to be difficult to pass air through. - Air transport. Collect air instead to be filtered later instead of filtering on the balloon. Drawback is that this method will be very labor intensive, no solution exists, and the equipment would still be difficult to transport. SAMPLE DESIGN Replicating the study enough times (both globally and in the same location) to build a substantial metagenomic (see Wooley et al. for an overview) dataset of cloud biodiversity over space and time. ENVIRONMENTAL DATA coming.. Importance of metadata Combing sampling with the environmental sampling technologies developed in EcoHackII to record the environment of our sample. SAMPLING SCHEMA location and replications per location (e.g. 10 samples in NYC), coming... FUTURE DIRECTIONS We intend to seek funding for continued sampling events and to cover the cost of continued sequencing. Funding will be used to ship the sampling kit to groups across the globe interested in replicating the study (classrooms, science events, and others). Ideally, this funding will be gathered through a combination of agency support (i.e. UNEP) and a microfunding campaign through a site such as Kickstarter or Petridish. Funding a travelling microbial sampling kit has valuable implications for scientific research, education, public health, and human safety. Knowing which microbes dominate the biodiversity in clouds over Manhattan, versus those in the skies over a city such as Dubai will help us better understand the links between microbes and air quality, climate, and disease. While the primary goal of the travelling kit will be new scientific knowledge, we will not be directly pursuing a publication from the data. Instead, data will be made available immediately following collection to anyone interested, likely including some of our partners. In this way, the citizens collecting the data can help advance a broad diversity of scientific missions. The project will represent many firsts in microbiology, aeromicrobiology, citizen science, and global biology research. PREVIOUS SAMPLING METHODS Bauer et al., Cultivable airborne microorganisms were collected directly from the ambient air under out-of-cloud conditions onto sterile cellulose nitrate filters (Sartorius, 47 mm ∅, bacteria: pore width: 0.45 μm; fungi: pore width: 0.8 μm), which were mounted in open face polycarbonate filter holders, which have been disinfected with 70% ethanol and isopropanol prior to sampling. The sampling time was 5 min at a flow rate of 24 l min−1. Loaded filters were placed onto agar plates within 10 min. SCIENTIFIC PUBLICATIONS Amato P. 2012. Clouds Provide Atmospheric Oases for Microbes. Microbe Bauer, H., A. Kasper-Giebl, M. Löflund, H. Giebl, R. Hitzenberger, F. Zibuschka, and H. Puxbaum. 2002. The contribution of bacteria and fungal spores to the organic carbon content of cloud water, precipitation and aerosols. Atmos. Res. 64:109-119. Wooley JC, Godzik A, Friedberg I (2010) A Primer on Metagenomics. PLoS Comput Biol 6(2): e1000667. doi:10.1371/journal.pcbi.1000667...