Mission 7 How is the Growth of the Bacteria Rhizobium radiobacter Affected by Microgravity? Montachusett Regional Vocational Technical School Fitchburg, Massachusetts

Team Members Marina Good: Principal Investigator Jezrielle Bruno: Collaborator Teacher Facilitator - Paula deDiego, Monty Tech

The Proposal The experiment was focused on how the growth of the bacterium, Rhizobium radiobacter, and by extension, the production of endotoxins, could be affected by microgravity, as opposed to its observed growth and endotoxin production on Earth.

Our Predictions Freeze-dried Rhizobium radiobacter can be successfully activated and grown in microgravity. More bacterial growth and higher endotoxin production will occur as a result of microgravity.

Our Packaging and Interactions - Volume 1: Freeze-dried bacteria - 1 ampoule of R. radiobacter - Volume 2: Growth Media - Filled to capacity with Luria Broth (LB) - Volume 3: Growth Inhibitor - 1 mL of 0.3 M Acetosyringone Interactions: U-5 [May 7th, 2016] - green clamp opened (activated experiment), inverted for 30 sec, mixing bacteria and growth media. U-2 [May 9th, 2016] - blue clamp opened (deactivated experiment), inverted tube for 30 sec, mixing growth inhibitor.

Analyses Performed Optical Density Test Supernatant Microscopy Analysis Mass Pellet Test Endotoxin LAL Biological Assay Extraction from cell membrane Supernate

Results for Optical Density ~ Supernatant Optical Densities Procedure 1 mL of each sample was taken, serially diluted 6 times (⅙ dilution), and analyzed at 600 nm, each cuvette containing 900 microliters of liquid. *Note: The serial dilution was made in triplicate. Results were recorded at dilution (10⁻¹) with 900 microliters of liquid in the cuvette. Ground Truth Tubes OD’s Flown Tubes GA 0.384 SA 0.372 GB 0.381 SB 0.392 GC 0.394 SC 0.406 Average OD of Ground 0.386 Average OD of Flown 0.39 This means that, based on these observations, there were virtually the same amount of cells (and growth) present in both samples, although the Flown Samples had a slightly higher average OD (0.39).

Microscopy of R. radiobacter Procedure: Both samples were streaked on slides, stained red (gram negative), and viewed underneath a microscope. Observations: The bacterial colonies of the Ground Truth were more compact, while those of the Flown Sample appeared slightly more spread out within their groupings. The cells of both samples were rod-shaped, as expected of a bacillus bacteria, but the cells of the Flown Sample appeared slightly rounded, while the cells of the Ground Truth were more distinctly rod-shaped.

Results for Mass Pellet Tube(1 mL per tube) Empty Tube (g) Tube w/ Pellet (g) Pellet Mass (g) Average Mass (g) G1 0.9849 g 0.9967 g 0.0118 g G2 0.9883 g 1.0006 g 0.0123 g G3 1.0168 g 1.0285 g 0.0117 g 0.01726 g G4 0.9947 g 1.0062 g 0.0115 g G5 1.0112 g 1.0502 g 0.039 g S1 1.0044 g 1.0149 g 0.0105 g S2 0.9852 g 0.9975 g S3 1.0060 g 1.0194 g 0.0134 g 0.01244 g S4 1.0014 g 1.0127 g 0.0113 g S5 0.9992 g 1.0139 g 0.0147 g 7 mL of each sample were spun in 14 microcentrifuge tubes at 14,000 RPM for 15 minutes. The supernatant was drawn off, and the masses of each of the remaining pellets of cell debris were taken. Key: G - Ground Truth [Ground] S - Flown Sample [Space]

Biological Endotoxin Assay for R. radiobacter The presence of endotoxins was confirmed after appropriate extraction from, and preparation of, both samples was complete, as well as an Optical Density Test, run at 545 nm. The high detection test was used, meaning that samples with added chemicals were incubated for 7 minutes (Time 2 [T2]) as indicated by the manufacturer. It was assumed that because of the cloudiness of the samples, there were large amounts of bacteria, resulting in high levels of endotoxins, requiring the high detection test, as opposed to the more sensitive low detection test (Time 1[T1]), used for lower concentrations of endotoxins. *Note: The Limulus Amebocyte Lysate (LAL) was slightly cloudy, so it was swirled for more than 30 seconds. **Note: The endotoxin was fully dissolved.

Results for Optical Density ~ Endotoxin Assay Extracted Endotoxins The high detection test was used, meaning that samples (with added chemicals) were incubated for 7 minutes (Time 2 [T2]) as indicated by the manufacturer. Samples were run at 545 nm, with 1 mL of liquid in each well. Samples were diluted (1 x 10⁻¹) 100 microliters of sample (supernatant) in 900 microliters of water (H₂O)

Optical Density Data Table ~ Endotoxin Assay Standards: Concentration Avg. Optical Density Minus Blank Blank 0.113 (Minus Blank Value) 1 EU/mL 1.084666667 0.9716666667 0.5 EU/mL 0.765 0.652 0.25 EU/mL 0.643 0.5303 0.1 EU/mL 0.42 0.307 *Note: EU/mL means endotoxin unit per milliliter. Sample Average Optical Density Subtracted Blank OD (0.113) Flown Supernatant (Diluted [10⁻¹]) 1.266333333 1.153333333 Flown Sample (Undiluted [10⁰] - Extracted) 1.212 1.099 Ground Truth Supernatant 1.235666667 1.122666667 Ground Truth 1.169 1.056

Results for Optical Density ~ Endotoxin Assay Sample Endotoxin Concentration (EU/mL) Ground Truth (Undiluted [10⁰] - Extracted) 0.275 EU/mL Ground Truth Supernatant (Diluted [10⁻¹]) 0.293 EU/mL Flown Sample (Undiluted [10⁰] - Extracted) 0.287 EU/mL Flown Supernatant (Diluted [10⁻¹]) 0.302 EU/mL *Note: EU/mL stands for Endotoxin Unit per milliliter.

Benefit of Knowledge Gained The Optical Density of the Flown Supernatant was slightly higher than that of the Ground Truth Supernatant, but not remarkably so. The bacterial colonies of the Flown Sample were not as compactly grouped as those of the Ground Truth. The bacteria of the Flown Sample appeared slightly rounded in shape, while the bacteria of the Ground Truth were more distinctly rod-shaped. The mass of the Flown Sample pellet was slightly lower than that of the Ground Truth pellet, but not remarkably so. The endotoxin concentration of the Flown Sample (of both the diluted supernatant and the undiluted sample extracted from the membrane) was higher than that of the Ground Truth (in both the diluted supernatant and undiluted sample extracted from the membrane).

Future Questions How is the structure and appearance of the cell membrane of Rhizobium radiobacter affected by microgravity? If samples of Rhizobium radiobacter were grown in space, returned to Earth, and continued to be grown, would the endotoxin production differ compared to a sample grown continuously on Earth? Would the genetic modification in plants, caused by Rhizobium radiobacter, be different if the bacteria was grown in space? As a commonly used vector in genetic modification and engineering in plants, would Rhizobium radiobacter grown in space be more efficient, less efficient, or even alter the nature of the introduction of a foreign gene?

Mentors ~ Thank you! Dr. Kristen DeAngelis University of Massachusetts Amherst Microbiology lab Doctoral Candidate Gina Chaput University of Massachusett Amherst Microbiology lab

North Central Community Foundation -Debbie & Phillips Richards Sponsors North Central Community Foundation -Debbie & Phillips Richards

Questions?