The purpose of experimentation was to determine which carbon source – glucose, glycerol, or maple syrup – caused Bacillus megaterium to produce the most poly-β-hydroxybutyric acid (PHB) by utilizing a UV spectrophotometer to compare the differing absorbance values of the bacterial byproducts at 235 nm and by employing a standard curve to calculate the dry weight of PHB in each media. One of the ultimate goals of experimentation was to ascertain an inexpensive method to produce great quantities of PHB, which can be used to create biodegradable plastics. Each of the carbon sources chosen had a specific advantage. Glycerol is an undesirable byproduct of methanol and alkali catalysis, a methyl ester process used to make biodiesel. Glucose is an easily accessible simple sugar, and maple syrup is a plentiful natural product. Thus, PHB production would be beneficial in any one of the media, but the most advantageous result would be if the greatest production was in the glycerol media so that the process of creating biodiesel would have minimal negative side effects. Production of PHB in glucose would be beneficial as well due to its accessibility, and maple syrup provides a natural process of generating a nature-friendly plastic. The study demonstrates that the most effective carbon source was maple syrup for the media containing maple syrup as a nutrient supplement led to the greatest production of PHB.

Bacterial Growth

Bacillus megaterium was cultivated in media that was prepared using sodium phosphate dibasic, potassium phosphate monobasic, sodium chloride, and ammonium chloride. The resultant broth was divided into four flasks, and a different carbon source – maple syrup, glycerol, and glucose – was added to each medium except the control in order to vary the nutrients each culture received. The bacteria were then transferred into the broth and left to grow for a week. During this period, a second trial was begun in which the media had more of each carbon source to determine if merely an increase in the carbon source was enough to cause the bacteria to produce more PHB. Therefore, the carbon to nitrogen ratio was raised to 6:1. The third trial involved media with nutrients derived from Tryptone Soya broth powder and then the various carbon sources added later on, which would indicate if excess carbon sources as well as normal nutrients would cause increased PHB synthesis (Fig. 1). Blanks of the broths in the second and third trials were created so subsequent growth in the broth could be detected in a UV spectrophotometer. In order to speed the rate of their growth, the bacteria in rounds two and three were incubated at 38 °C for 24 hours. After 26 and 72 hours, the bacteria were scanned in the spectrometer to see the amount of growth over time.

PHB Detection

In order to create PHB suspensions, a vortexed sample of each broth was extracted and centrifuged to isolate the bacteria (Fig. 2). The cell paste was resuspended in a sodium hypochlorite solution for 1 hour at 37 °C, which lyses the bacteria’s cell walls. After being washed by water, acetone, and ethanol, the remaining lipid granules were transferred into boiling chloroform, which extracts the PHB and dissolves any residual contaminants using the set-up (Fig. 3).

After five minutes, the chloroform extract was filtered through 42.5 mm filter paper and dried on a hot plate. 10 mL of 99% sulfuric acid was then added to the extract, and this mixture was heated and stirred, converting PHB to crotonic acid (Fig. 4). This solution was compared against a sulfuric acid blank in the UV spectrophotometer at 235 nm. A standard assay was created, enabling the amount of PHB produced to be determined based on the absorbance at 235 nm.

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