Determining presence of microbial communities using high altitude balloons

Research Group: Taylor Burchard, Carcie Chappell, and Jericho Simone

Launch: Whitworth Spring 2020

''For decades, one of the most unexplored environments of Earth has been the atmosphere. The biodiversity found is vastly underexplored and very rarely researched. During this semester of the Near Space Research program, we wanted to attempt to test the quantities of microbial communities found at different levels of the atmosphere. This way, we could gain a better understanding of the number of biological species able to survive in such extreme environments. The design of this experiment was based around a capsule that would collect samples at different altitudes. The capsule would contain vials on a rotating platform that would shift places in accordance with a timer. As the vials shifted, one would be in an opening where it would have a fan that would pull air through the vial thereby collecting a sampling of that altitude onto the filter paper in the vial. While we were not able to test this design and collect data due to the COVID-19 pandemic, our designs and researched contained in this wiki could help another group in the future research this same topic.''

Background
In many ways, the atmosphere is one of the most unexplored parts of Earth’s ecosystem. In many ways, there is an entire microbial ecosystem in the stratosphere. There has been previous literature on how many microbes are present in the lower atmosphere near the ground level. They found that at lower altitudes, there was a large variety of microbes present in both types and quantities. However, high altitudes are far less explored. For decades, scientists have tried sending balloons up into the stratosphere to attempt to detect microbes. The first attempt was over 75 years ago and since then almost every study has been based on simply detecting the presence of microorganisms. In 2014, a group out of Louisiana State University created a high altitude balloon that could travel up into the atmosphere and capture samples of different bioaerosols. While they did not actually test that particular contraption, different groups have expanded on the idea and sampling microorganisms in the atmosphere has become ever more prevalent.

NASA has been sending up aircrafts multiple times to try and detect the types and amounts of bacteria present. They found that at the lower levels of the troposphere there are still microorganisms present. In fact, they found that these microorganisms could have an effect in directing the weather by the process of accumulation. Basically, once enough of the microbes form together to create their own microbiomes they begin to attract water droplets and soon clouds can form which will affect the weather nearby. These are extremely hardy types of bacteria, able to survive extreme conditions and these extremophiles are likely what we would find in the atmosphere of other plants. Just two years ago in 2018, NASA sent up another high altitude balloon that took collections of samples up to 13.7 km into the atmosphere.

However, they only went ahead to detect the kinds of bacteria found in the lower stratosphere. They found five different taxa in the stratosphere. Noticeably, these five taxa were also found in the lower altitude samples also taken. This suggests that microbiomes can extend into multiple elevations and that these extremophiles are far hardier than originally thought. While they did measure the number of bacteria in the stratosphere this NASA flight has so far been the only one taken of its kind and was far more interested in the type of bacteria than the amount. They only have a rough estimate for the number of microbes found in the atmosphere.

Theory
We want to collect bacteria at different altitudes with an active filtration process. We will use a helium balloon which reaches a max altitude of about 37 km. The composition and prevalence of microorganisms in the middle-to-upper troposphere is around 8–15 km. The concentration of microbial cells is typically 0.1–3 μm in diameter, in the lower troposphere (<5 km altitude), which is how we determined which filter to use. To collect microorganisms in the troposphere and low stratosphere, two filter samples are taken at four different altitudes with polycarbonate micron filters. Two of the eight samples will be taken at ground level for control. A system including a fan, which actively transports material from the outer atmosphere into a designated tube and onto a polycarbonate micron filter, will allow collection from the exterior surroundings in the atmosphere to inside the enclosed capsule.



The experiment will be attached to the tail of a helium balloon and the balloon will reach a max altitude of 30 km at a constant velocity until it descends. Eight total filter samples will be taken at chosen altitudes. We can further analyze the filters collected in the lab through serial dilutions and culturing. We expect to find a larger abundance of microorganisms sampled at lower altitudes since bacteria travel into the atmosphere by dust, water, human pollutants, and/or jet streams. The lower the altitude, the larger the number of bacteria we will find. As the altitude increases, there will be fewer. The capsule will spin accordingly, exposing one tube with a filter, at a specific time, to the atmosphere. We will do this by designing a capsule in which keeps samples isolated from the exterior until rotated by command.

We want to measure the number of bacteria at different altitudes as well as diversity. The filter samples will be added to a buffer solution, to maintain a 7.4 pH to keep the samples alive. We will perform serial dilutions to dilute the original solution containing the filter samples of bacteria by a ten-fold decrease dilution factor. Then, we will grow the bacterial samples by culturing them from the test tube solution onto an agar plate. Taking a small volume of solution out of a test tube sample and placing it onto the growth medium allows us to quantify the bacteria. The quantity can be determined by calculations which can be found in the Validation and Calibration section. We then perform the appropriate calculations of CFU's, or colony-forming units, to determine the original amount of bacteria present in the original solution.

We would determine the diversity by looking at the cultures, where we will observe the different phenotypes of different colonies. When the experiment is back onto the ground and detached from the helium balloon, the filters will be taken to the laboratory for further analysis. We will place the filters into a buffer solution of phosphate buffer saline since it is good for biological life and has a 7.4 pH. If we separate each filter and analyze it separately, we can compare the end results. Each filter will have its own separate process of serial dilutions. There will be eight separate test tube racks. Each dilution will decrease the number of microorganisms from the original solution in test tube one by a ten-fold. All individual test tubes will have their own agar plate for culturing. The solution from each test tube will be spread along the top of separate agar plates. By diluting the original solution in each test tube rack, we can quantify the microbial communities on the agar plates. Based on the number of colony-forming units, CFU's, represented on the plates, we will be able to calculate the number of microorganisms present in the original test.

Design
Our design section has four main parts: mechanical, electrical, software, and procedural design. Since our collection process contained active filtration, the capsule, which was 3D printed (see below for 3D print instructions), was what would contain all the parts to our experiment. The filters, the fan, and the samples collected in the atmosphere. The mechanical design explains how to assemble the capsule in which our collection process takes place. The software and electrical are for the timer and/or our flight computer in which tells the disk within the capsule when to rotate to expose a section of the disk to the outer atmosphere. Here you will find our code flow and information about the servo, which is what allows for the disk to turn. The bulk of the experiment will be carried out after the launch date. The cultures take at least 24 hours to grow, therefore, do not wait until the last minute to perform the analysis.

For Future Reference:

Before launch day, we would recommend collecting samples at ground-level, early on the semester. Perform serial dilutions and cultures in labs with ground-level samples to test that you know how to do this properly. Since you only get one analysis with the data from the atmosphere, it is important to be prepared. You can record this ground data as your control and keep the data to compare it to the atmospheric data. Also, before launch day, set up the lab ahead of time so that you can get started right away once you return.

Our collection process for filtration could have been simpler. We spent quite a bit of time figuring out how to collect microbes in the atmosphere, and 3D printing, when we could have been doing trial runs. There are multiple ways to collect samples. Using the nylon membrane filters is important because they are the right pore size to collect microbes, but there are active and passive filtration processes. An active filtration process is where we facilitate the sample going through the filter. One example of this would be a fan pulling air through the vial. This is the process we ended up choosing; it would be the likeliest to give us the largest sampling of microbes as it would pull in more air and therefore more sample. The other option we did not take was passive filtration. As the name implies, this is where there are no fancy bells and whistles to pull the air through, and instead, the system relies on what would occur naturally. One example of this would be the impact method where the vials are simply exposed to the air and it relies on the air impacting the filter with enough force to allow the microbes to stay.

We did not get to carry out this experiment, nor do prior testing in the lab.

Mechanical
Be wary of cross-contamination with the outer air. Assemble filters in a sterile environment, such as under a fume hood, and wear gloves.

The outer dimension of the capsule is a hollow cylinder with a radius of 6cm and a height of 7.75cm. The width is 0.5cm (see figure 4). The cap for the hollow cylinder will have a lip height of 0.5 cm with an inset of 0.5cm, for a total height of 1cm. With the cap, the inset has a radius of 5.75cm. The bottom of the capsule and the cap will have eight holes along the outer perimeter, of a diameter of 1cm. An inner plate/disk will be on an axis (which is connected to a servo) within the capsule, which contains the eight holes (see figure 2). The holes are spaced equally apart and open into the inner space of the capsule. The plate will hold our sample tubes. The tubes will be set into the plate, with a lid on one end of the tube to create a semi-isolated environment. The lid will be held in place by the end cap of the capsule and will spring open when exposed to the holes in the caps. Both centers of the holes in the caps and the center of the tubes will be 5cm away from the center point of the capsule axis. Tape will secure the closing of the capsule by sealing the cap. A filter will be placed on the ends of the tubes. The filter will collect microbes at specific altitudes based on time. This is an active collection method and fans will be placed on the bottom of the openings on the filter. This will move the microbes from the atmosphere to the inside of our experiment through a slot in our capsule. At the bottom of the capsule is a fan that is pulling in air from the outside. What this means is that the fan creates that system of active filtration where the samples can be pulled into their respective vials and into the filters. This way, the fan makes sure we get at least some samples no matter the altitude.







The capsule and its accessories were 3D printed.   

Electrical
Our electrical system is very simple, it contains the flight computer with its components and one continuous-rotation servo. The servo would be connected to ground(p1), PWMout(p24), and power(p2). All other connections, such as ground and power, are already made within the flight computer.

Software
Six samples will be collected in the atmosphere at chosen altitudes based on time. Every ____ seconds the disk will turn, exposing a filter to the atmosphere. 

Collecting and Culturing Procedures
Launch Day Note: The GPS attached to the helium balloon has already been set up. Helium, tank, and pump already provided. Once the balloon has landed and is found, take the box and put the samples into a cooler to take back to the lab. The cooler should be at a temperature of around 37 degrees celsius.

Making Phosphate-Buffered-Saline
This buffer solution will be used as the solution that the filters containing bacteria will be placed in. This is the only fluid used in this procedure. (pH = pKa + ln [A^_]/[HA^+]) A buffer is a weak acid/weak base. Phosphate-buffered-solution is isotonic and non-toxic to cells, good for biological research. Have this set-aside.
 * 1) Prepare 800 mL of distilled water in a suitable container
 * 2) Add 8 g of NaCl to the solution
 * 3) Add 200 mg of KCl to the solution.
 * 4) Add 1.44 g of Na2HPO4 to the solution. base
 * 5) Add 240 mg of KH2PO4 to the solution. acid
 * 6) Adjust solution to desired pH (typically pH ≈ 7.4).
 * 7) Add distilled water until volume is 1 L.

Conducting the Lab Experiment
This is when the analysis of the bacterial filter samples will begin. Before any dishware is used, it must be sterilized to remove anything that may interfere with accurate data collection.
 * 1) Sterilize all tubes using autoclave 121%, 15 psi for 20 minutes
 * 2) Dip tool into acetone and then place in a burner for a couple of seconds, then take it out and it is ready for use

Preparation of Test Tubes
The test tubes will contain PBS and the filter samples. Organize the test tubes in the test tube racks. There should be eight test tube racks total since there are eight filter samples. Each filter sample is to be analyzed separately, in its own series of dilutions. PBS should be made already. The number of test tubes may vary in different racks, as well as the volume of buffer solution. We did it this way since the abundance of bacteria in the atmosphere decrease with altitude. With calculations, we determined that if the higher altitude samples were diluted to the same factor as the lower altitude samples, then either the dilutions would be too dense for the lower altitude samples, or the dilutions would be too diluted for the higher altitude samples. The CFU's on the plate of analysis must be between 30-300 CFU's. If the count is higher than 300, it is nearly impossible to count. If the count is lower than 30, it is not enough data to form a conclusion.
 * 1) In a large volumetric flask, prepare 200.0 mL of phosphate-buffered saline (PBS)
 * 2) Gather eight test tube racks
 * 3) Place five test tubes in two of the racks. Add 10mL of PBS to the first test tube in both of the racks. Add 9.0 mL of PBS to the remaining four test tubes in both of the racks.
 * 4) Add four test tubes to the other six racks. Add 5.0 mL of PBS to the first test tube in all four racks. Add 4.5 mL of PBS to the three remaining test tubes per rack.
 * 5) There are eight filters. Two per chosen altitude (refer to?). The two filters at ground level are tested using larger volumes of PBS since there is a larger abundance of microorganisms at lower altitudes. (Refer to calculations). The other six filters are tested with smaller volumes of PBS since they increase in altitude over time with the balloon.
 * 6) Clearly label the test-tube racks as well as the test-tubes with the altitude and associated filter

Ground Level Altitude Samples
You should see growth within a couple of days. The dishes will start to smell which means the bacteria are growing. Make observations and keep records of what you see growing in each dish. Before disposing of dishes in the trash the bacteria should be destroyed. Pour a small amount of household bleach over the colonies while holding dish over the sink. Caution - Do not allow the bleach to touch your skin, eyes, or clothes.
 * 1) For rack one: Take one of the filters from ground level and place it into the first test tube containing 10.0 mL of PBS
 * 2) Pipet 1.0 mL of solution from test tube one into test tube two. Total volume in test tube two: 10mL.
 * 3) Pipette 1.0 mL of solution from test tube two and place it into test tube three. Repeat serially for the remaining test tubes.
 * 4) Cap test tubes along the way and vortex to mix well.
 * 5) The first test-tube has a dilution ratio of 1:1, the second 1:100, the third 1:1000, etc. The serial dilutions decrease the density of the original solution by a 10 fold factor. This allows for an easier calculation of microbial colonies. Once the solution from the test-tube is transferred to an agar plate, the test-tube with a larger dilution factor will have fewer microorganisms on the agar plate. This helps to quantify the amount on one plate and allows for an easier calculation of microbial communities.
 * 6) Gather agar plates: one per test-tube.
 * 7) Using a pipet and disposable tips, pipet 0.1 mL of the solution from test-tube one onto agar plate one
 * 8) Dip an L-shaped glass spreader into ethanol
 * 9) Flame the glass spreader over a bunsen burner to sterilize
 * 10) Once cooled, dip into the test tube with the bacterial sample
 * 11) Spread the sample evenly over the surface of the agar plate in a streaking or S pattern. Spread the loop across the surface about four times. Retrace this pattern four times more, flaming the spreader in between
 * 12) Repeat this process for the remainder of the test-tubes in rack one. Repeat process for the ground filter #2 rack.
 * 13) Cover the petri dish with parafilm and label it with its dilution factor, altitude, and sample number
 * 14) Let it grow. Incubate the plate inverted—with the agar-containing part of the plate up—at 37°C overnight or for 24 hours.



High-Atmosphere Altitude Samples


You should see growth within a couple of days. The dishes will start to smell which means the bacteria are growing. Make observations and keep records of what you see growing in each dish. Before disposing of dishes in the trash the bacteria should be destroyed. Pour a small amount of household bleach over the colonies while holding dish over the sink. Caution - Do not allow the bleach to touch your skin, eyes or clothes.
 * 1) Gather racks and test-tubes for the high-altitude filter samples.
 * 2) Prepare 5.0 mL of PBS and place it into test-tube #1. Add one filter.
 * 3) Cap test-tube and vortex to mix well
 * 4) Pipet 0.5 mL of test tube one into test tube two. The total volume of test-tube two: 5.0 mL
 * 5) Cap test-tube and vortex to mix well
 * 6) Repeat serially for the remaining test-tubes.
 * 7) The first test-tube has a dilution ratio of 1:1, the second 1:100 ratio, the third 1:1000, etc
 * 8) Using a pipet and disposable tips, pipet 0.1 mL of the solution from test-tube one onto agar plate one
 * 9) Dip an L-shaped glass spreader into ethanol
 * 10) Flame the glass spreader over a bunsen burner
 * 11) Use the spread example: spread the sample evenly over the surface of the agar plate
 * 12) Rotate the petri dish at an angle of 45 degrees simultaneously
 * 13) Cover the petri dish and label it with its dilution factor, altitude, and sample number
 * 14) Repeat this process for the other test-tubes in each rack, as well as the other test tube racks taken at higher altitudes
 * 15) Cover the petri dish with parafilm and label it with its dilution factor, altitude, and sample number
 * 16) Let it grow. Incubate the plate inverted—with the agar-containing part of the plate up—at 37°C overnight or for 24 hours.

Post-Culturing
See equations in VALIDATION AND CALIBRATION section
 * 1) Gather plates
 * 2) Determine the countable plate (between 30 and 300 colonies). There will/may be plates too numerous to count (TNTC). >300 colonies would be difficult to count, and <30 colonies are too small a sample size to present an accurate representation of the original sample
 * 3) Repeat for each rack of test tubes
 * 4) Calculate the number of CFU's on the countable plate, the number of microorganisms per mL. (INSD)
 * 5) Calculate how much the original sample was a diluted total (TNSD). *Do NOT include any dilutions after the countable plate.
 * 6) Calculate the dilution factor once the sample is plated (CFU/mL). (PDF)
 * 7) Calculate the final dilution factor (FDF). Tells you how much the original sample was concentrated.
 * 8) Calculate colony forming units (CFU/mL) in the original sample (all of the dilution of the original sample)

Servo
Our team did not receive our continuous rotation servo in time to work with it before the COVID-19 shutdown. Due to this, we were not able to calibrate or validate the rotation amounts that would be required for each turn of the plate within the capsule to expose and re-cover each sample. However, our team was able to gain some experience with servos by working with an mbed and a limited rotation servo. This was done by using code that would rotate the servo to a certain degree, and then illuminating one of the mbed light, then repeating the process with a further rotation and an additional light. This process of rotating to a given degree and then turning on an mbed light would have also been used for the continuous rotation servo, had the opportunity been presented. This would give us information on how long the pulse would need to be for our desired rotation while taking account any margin of error in the particular servo we would have had.

After setting up the system of the capsule, plate, and servo, the validation of the servo rotation would've been done visually. This would be done by simply running the code and confirming that each sample lined up with the opening in the capsule lid, and then closed off each sample between collection points.

Calibration
There was no opportunity to do calibrations for the servo or the code as neither existed but we did create ways to calibrate the serial dilutions and know how to calibrate the biological procedure in order to make it work.

Calculations for Serial Dilutions
Once agar plates have been incubated for 24 hours, visual analysis occurs. To determine the number of bacteria in the first test tube on each test tube rack, at least one agar plate from each rack must proceed through this series of calculations. Since we know the ratio in which the original test tube sample was diluted, the amount of volume added to each test tube, the volume added to each plate, and the number of CFU's on at least one plate, the amount of bacteria in test tube #1 can be determined.

Dilution Factor (DF)
The dilution factor is the amount each test tube is diluted by. To solve for the dilution factor, divide the amount of initial, or added, volume by the amount of final, or total, volume. Below is an example where the dilution factor is 1/10. The dilution will keep decreasing by a factor of 1/10 serially. For example: If 10mL/10mL=1, then the dilution factor is 1mL/10mL=1/10x. Calculating for the consecutive test tube would be 1mL/10mL=(1/10)(1/10)=1/100 and for the next, 1mL/10mL=(1/10)(1/100)=1/1,000, etc.
 * 1) (DF)=(v_i)/(v_f)

Individual Serial Dilution (INSD)
Calculate the number of CFU's on the countable plate, the number of microorganisms per mL. (INSD)
 * 1) (INSD)=Number of colonies/dilution factor

Total Series Dilution Factor (TSDF)
Calculate how much the original sample was diluted total (TNSD). *Do NOT include any dilutions after the countable plate. The total series dilution factor is a calculation of how much the sample was diluted in all of the tubes combined. This is accomplished by multiplying each of the appropriate INSD. This series does not include any dilutions after the countable plate.
 * 1) TNSD=INSDx

Plated Dilution Factor (PDF)
PDF = mL plated/1 mL Calculate the dilution factor once the sample is plated (CFU/mL).

Final Dilution Factor (FDF)
Calculate the final dilution factor (FDF). Tells you how much the original sample was concentrated.
 * 1) FDF = SDF x TSDF x PDF

Colony Forming Units (CFU/mL)
To find how many bacteria were in the original sample.
 * 1) CFU/mL = (CFU's on the countable plate) x (1/FDF)

Data and Analysis
Due to the COVID-19 pandemic, this experiment was never completed.

Conclusion
In the end, this ended up being a mostly theoretical venture. We did not have the time to complete test runs of our system and while we have a completed procedure we did not get to use it. Overall, if another group wants to continue where we left off and keep on working on the mechanical and electrical work then we have a strong foundation and base. The biology portion of this is complete so all it would take is an intrepid mechanical engineer to continue the project where we left off. While the pandemic stopped us in our tracks, the outline and foundation for a unique and interesting exploration into the atmosphere are still present. We hope one day someone might pick up this project where we left off.