Does Bacteria Growth in Milk Happen Differently in Microgravity than It Does on Earth ?

1. Mammalian Milk in Microgravity Does Bacteria Growth in Milk Happen Differently in Microgravity than It Does on Earth? NEST+m 5th Grade Student Spaceflight Experiment Design

2. Mammalian Milk in Microgravity NEST+m 5th Grade Student Spaceflight Experiment Design Team Principal Investigators: Lennie H Ma & Xander Plössl Teacher Facilitators: Zach Vine & Marvin Cadornigara Coordinator: Brendan Alfieri Collaborators: Maury Ahram, Kiara Bracero, Clarisa Carrillo, Kaylyne Cruz, Laila Cruz, Sebastian Delangle, Joseph Dell'Olio, RemmiDuplessis, Mina Ekstrom, KunalGahlawat, Kathryn Galperin, ZubinGell, Kamaya Gooding, Theo Haegele, Dawson Hall, Hayley He, Elliot Leinweber, Cavan Miller, Georgia Podgainy, Nia Powell, Samantha Rapkiewicz, Sasha Roberts, Julien Saunier, Felix Scaggiante, Isabella Serrano, & Riley Sexton Does Bacteria Growth in Milk Happen Differently in Microgravity than It Does on Earth?

3. Experimental Background • Microorganisms play important roles in the quality and safety of dairy products • Bacteria are probably the most important group of microorganisms in dairy • Pasteurization and powdered milk production does not kill all the bacteria • Spoilage in space can cause significant problems • A new or better understanding of how milk and other foods spoil in microgravity might allow people to colonize different places in space

4. Experimental Proposal • Does Bacteria Growth in Milk Happen Differently in Microgravity than It Does on Earth? • Hypothesis • Milk will grow and host bacteria differently in microgravity than it will on Earth • A microgravity milk sample will grow fewer bacteria than the ground truth experiment milk sample • Proposal • Compare bacterial growth and content of a milk sample in microgravity to milk samples in full gravity • Use standard methods of bacterial examination in milk to compare samples • Work with powdered milk and distilled water to limit the types and amount of bacterial growth before activation • Spore forming heat thriving (thermophilic) and heat resistant (thermoduric) bacteria

5. Experimental Design • Fluids Mixing Enclosure • Type 2 FME • 6 ml of distilled water • 1.5 ml of powdered milk • Bacterial Examination • Standard Plate Count • Culture dilute solutions of samples on agar • Count colony forming units (CFU) and calculate bacteria concentrations • Reductase Test • Use methylene blue to indicate the absence of oxygen due to bacterial growth

6. Results • Observations • Flight Sample • Returned to Earth intact but seal broke (blow out plug) between return and laboratory examination • Liquid was visible in container bag • FME milk sample was dark brown and appeared burnt • Ground Samples • FME seals were intact • FME milk sample was milky white • Bacterial Examination • Standard Plate Count • Flight Sample averaged 260 CFU/ml milk • Ground Samples were greater than 10,000CFU/ml milk • Reductase Test • Ground Sample took more than 8 hours to change color, indicating a low rate of oxygen depletion • Flight Reductase Test inconclusive because of dark coloration of sample • Use methylene blue to indicate the absence of oxygen due to bacterial growth

7. Results • Flight Sample • Returned to Earth intact but seal broke (blow out plug) between return and laboratory examination • Liquid was visible in container bag • FME milk sample was milky brown and appeared burnt

8. Analysis • Flight Sample • Likely causes of FME failure was gas pressure build up from continued growth of bacteria • Delays between Flight Sample return and laboratory testing contributed to failure • Only limited conclusions possible because of FME failure • Low Standard Plate Count may have been the result of die off • Ground Truth Samples • Expected, high bacterial growth rates were recorded • Results validated basic experimental procedures • Comparison • Flight and Ground Truth Samples could not be directly compared because of Flight FME failure • The Flight FME showed likely had much higher bacterial growth as no similar gas build up and failure occurred in any of the Ground Truth Samples

9. Conclusions & Recommendations • Conclusions • We could not conclude that the Flight Sample had a higher or lower rate of bacterial growth than the Ground Truth Samples • We can conclude that there was a greater gas production in the Flight Sample which may indicate a greater rate of bacterial growth • Recommendations • Experimental design should better control variables not being tested. Examples: • The period of bacterial growth should be shorter to limit variance • Halting bacterial growth before returning Flight Sample would make the sample handling more similar • Temperature is very important in bacterial growth and survival and needs to be the same between Flight and Ground Truth Samples • Sample examination and testing must be well organized and done quickly for biological experiments

10. Acknowledgements • The NEST+m Student Spaceflight Experiment Design Team would like to thank: • Local Partner organizations that made SSEP possible for the community • NY Space Grant Consortium (Cornell University) • Center for the Advancement of Science in Space • Step I Review Board • Marvin Cadornigara, Taehyung An, Richard Sullivan, Joanne Ristau, Katelin Corbett, Evan Feldman, Carla McGreggor, Danielle Neubauer, Hyungmin Park, Zachary Swenson & Richard Sullivan • Research Institutions for Sample Prep and Sample Testing Assistance • Hunter College, Department Of Biological Science • George Wallace, Chief College Laboratory Technician • New York University • Professor Edo Kussell, Mark Rocco, & Wei-Hsizng Lin, Center for Genomics and Systems Biology • Dr. David Greer. Department of Physics • Advisors • Dr. Graciela Ramirez Toro, Director of CECIA, InterAmerican University • Steven C. Murphy, Department of Food Science at Cornell University MILK IN SPACE

11. NEST+m Student Spaceflight Experiment Design Team