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Brookhaven Academy

Brookhaven Academy. 12th Grade. Polyhydroxyalkanoate Production in Microgravity. Flight Experiment Mission V to ISS. Co-principal Investigators: Garrett Smith Lindsey Winborne Samantha Barton Ashlea Bardwell Ruth Vaughan.

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Brookhaven Academy

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  1. Brookhaven Academy 12th Grade

  2. Polyhydroxyalkanoate Production in Microgravity Flight Experiment Mission V to ISS

  3. Co-principal Investigators:Garrett SmithLindsey WinborneSamantha BartonAshlea BardwellRuth Vaughan

  4. Teacher Facilitator:Dr. Dianne Watson Leslie Hood a.k.a. “Hood Momma”

  5. Special Thanks • Selby and Richard McRae foundation • Entergy foundation • MS space consortium • Brookhaven academy educational foundation • DrDonna Sullivan UMC

  6. Purpose • The purpose of the experiment is to determine if the bacteria, Ralstoniaeutropha, will create polyhydroxyalkanoate(PHA) after being exposed to microgravity for three weeks. PHA is a biodegradable polyester created by bacterial fermentation.

  7. The bacteria that will produce PHA in this experiment is Ralstoniaeutropha. The bacteria produces PHA through bacterial fermentation which is a process that breaks down the carbon source and nutrient broth leaving behind pellets of PHA.

  8. Materials • •Ralstoniaeutropha • •Phosphate Buffered Saline • •Brain Hearth Infusion Broth • •RNA later II • •Type 3 FME

  9. Procedure: • Ralstoniaeutrophawas provided by University of Mississippi Medical Center. Ralstoniaeutropha will be used to examine the production of PHA. • Screening of PHA producing microorganisms was carried out by Nile Blue Staining method (Ostle and Holt, 1982). Nile blue is dissolved in acetone, and was added to the agar medium for viable colony staining. PHA producing microorganisms were visualized as bright orange colonies under UV transilluminator (Spiekermann, 1999)

  10. The growth of SP-Y1 in production medium was monitored by taking absorbance at 660nm and the corresponding amount of PHA accumulated was determined by incubating it for five consecutive days (Henderson and Jones, 1997). • The stock cultures (R. eutropha and the isolates) are initially revived in nutrient broth andlater inoculated into mineral salts medium containing hydrolyzed grass (10g), glucose (5g), sodium chloride (5g), di-potassium hydrogen phosphate (5g), magnesium sulphate (1g) and ammonium sulphate (1g) in 1L-distilled water. The pH is adjusted to 7.4±0.05 and the cultures were incubated for 48h at 30oC in orbital shaker (Du et al., 2001; Amirul et al., 2008; Yamanaka, 2010

  11. After the incubation period of 48h, the cultures were centrifuged at 10,000rpm for 5min. The supernatant was discarded leaving the pellet, which was air dried and weighed. The extraction was done following the method of Santhanam and Sasidharan (2010), where PHA is extracted using the solvent chloroform. The cell pellet was suspended in sodium hypochlorite solution and incubated at 37°C for 1-2 hours for complete digestion of cell components except PHA. The mixture was centrifuged to collect PHA granules and the supernatant was discarded. The sediment was washed twice with distilled water and centrifuged again. Finally PHA granules in the sediment were washed twice with acetone and diethyl ether (1:1 ratio). (Aravind et al. 2012/13)

  12. The granules of PHA were identified by transmission electron microscopy, and then the bacterial cultures were centrifuged at 10,000 rpm for 5 minutes to obtain the cell pellet • With a sterile pipette, we loaded 2.5 ml of RNA Later II (or we could have used formalin) into mini-lab Volume 3. • With a sterile pipette, we loaded 2.0 mls of Brain Heart Infusion Broth into volume 2 of mini lab. • With a sterile pipette, we loaded .5 ml of Ralstoniaeutropha suspended in Phosphate Buffer Solution into volume 1 of the mini lab

  13. After the Flight • The University of Mississippi Medical Center will then perform transmission electron microscopy to determine the amount of PHA production.

  14. Hypothesis: • The experimental hypothesis is that more PHA granules will be produced in microgravity than on earth.

  15. We are very honored to represent the great state of Mississippi and our school, Brookhaven Academy, and to be the first from Mississippi to be a part of the SSEP.

  16. Thank You For Your Time

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