Brookhaven

Academy12th Grade

Polyhydroxyalkanoate Production in Microgravity

Flight Experiment Mission V to ISS

Co-principal Investigators:Garrett SmithLindsey WinborneSamantha BartonAshlea BardwellRuth Vaughan

Teacher Facilitator:Dr. Dianne

Watson Leslie Hood

a.k.a. “Hood

Momma”

Selby and Richard McRae foundation

Entergy foundation

MS space consortium

Brookhaven academy educational foundation

Dr Donna Sullivan UMC

Special Thanks

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

Purpose

The bacteria that will produce PHA in this experiment is Ralstonia eutropha. 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.

•Ralstonia eutropha

•Phosphate Buffered Saline

•Brain Hearth Infusion Broth

•RNA later II

•Type 3 FME

Materials

Ralstonia eutropha was provided by University of Mississippi Medical Center. Ralstonia eutropha 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)

Procedure:

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

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)

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 Ralstonia eutropha suspended in Phosphate Buffer Solution into volume 1 of the mini lab

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

After the Flight

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

Hypothesis:

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.

Thank You For Your

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