Bacterial Growth

1. Introduction 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 nmand 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. Fig. 1 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). Fig. 3 Fig. 2 Fig. 4 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.

2. Production of Poly-β-Hydroxybutyric Acid with B.megaterium in Carbon Sources Gretchen Sileo and Amy Sung Hopkinton High School Hopkinton, MA Chen-Lu Yang Advanced Technology and Manufacturing Center University of Massachusetts - Dartmouth Table 1 PHB Spectrophotometric Assay Results Fig. 5 Fig. 6 Table 2 Amount of PHB Present in Each Media  Fig. 7 Fig. 8 Table 3 Bacteria Growth in Media of Various Carbon Sources

3. Bacterial Projected Growth Phases Control Glucose Glycerol Maple Syrup Stationary Log Death Lag University of Massachusetts Dartmouth Chapter of Sigma Xi, the Scientific Research Society 15th Annual UMass Dartmouth Research Exhibition April 28-29, 2009 Discussion and Conclusions The bacteria in the maple syrup medium produced the most poly-ß-hydroxybutyric acid (PHB), corroborating the hypothesis. However, the reasoning behind the hypothesis was disproved, due to the observed patterns of growth of the bacteria in the various media. The Bacillus megaterium growing in the control medium, which only had the minimal medium and no added carbon sources, grew rapidly, with an initial average 0.736 AU absorbance to an average 1.267 AU. While the bacteria had sufficient nutrients to grow normally, the lack of significant production of PHB indicates that the excess carbon added to the other types of media prompts a type of regulatory mechanism within the bacteria that initiates the activity of PHB synthase. Because PHB synthase was not prompted, it could not catalyze the polymerization of PHB. Therefore, although the bacteria grew significantly, they did not produce a substantial amount of PHB, conveying how bacterial growth and PHB production are not always proportional. The concentration of bacteria in the glucose medium slightly decreased over the 45 hours in which the broths’ absorbance values were determined, even though the medium enabled the bacteria to produce 8.2 μg/mL of the polymer, the second highest amount out of all the types of broth. The regulatory mechanism detected the excess carbon and induced the enzyme to begin polymerization, but the 20% glucose solution that was originally added to make the broth only contained 1.8g of glucose. The bacteria, while in the log phase, rapidly utilized the glucose, but after the carbon source was significantly depleted, they entered the stationary phase prematurely and theoretically had a balance between new and dying cells (Fig. 9). However, since this equilibrium is ideal, the bacterial growth actually fluctuated slightly to allow for irregularities, which illustrates why the absorbance for the bacteria in glucose seems to have dwindled somewhat. Because the prokaryotes had just entered the stationary phase, polymer production appears to be elevated, since this is the point in the growth curve when PHB production is at its peak. Fig. 9 The growth of the bacteria in the glycerol medium only increased marginally, and only slight amounts of PHB were produced, mainly due to the chemical structure of glycerol. Glycerol (C3H5(OH)3) has hydrogen bonds and can form hydrogen bonds with water in an aqueous solution. Because the Tryptone Soya Broth powder had 250mL water added to it to create broth, glycerol created a network of hydrogen bonds that the bacteria could not break down easily to convert into food. After it used the glucose from the minimal medium to produce a small amount of PHB, it could not use most of the glycerol because of its inability to break down the hydrogen bonds and therefore, like glucose, entered the stationary phase early. The bacterial growth for the maple syrup was the most significant over a period of 45 hours, increasing from an average 0.539 AU absorbance to an average 1.531 AU absorbance, almost tripling (Fig. 10). Containing carbohydrates, minerals, phenolic compounds, and amino acids, the maple syrup, when added to the minimal medium which already provided a small amount of a carbon source and amino acids, allowed the bacteria to sustain itself in the exponential growth phase for a longer period of time and become more abundant. The amount of PHB that the bacteria in the maple syrup produced was also significantly higher than the other bacteria samples (19.80 μg/mL) because, not only did it have the excess of carbon source necessary for the regulatory mechanism to prompt PHB synthase to begin producing polymer, it also had the surplus of nutrients to maintain bacteria that were multiplying exponentially and all producing PHB for an extended period of time. Fig. 10 The results can be further explained by examining the role of phasins in PHB production. Once PHB synthesis is initiated, PhaR, a negative regulator, binds to nascent PHB granules and transcribes phaP, a gene that produces phasins, or granule-associated proteins that collects on the outside of the granules and prevents them from coalescing as they grow larger (Fig. 11). This regulates the surface-to-volume ratio and thus promotes the production of PHB. While the presence of PHB results in increased synthesis of phasins, the presence of phasins helps produce more PHB, resulting in a large concentration of PHB when phasins are being transcribed. At the later stages of PHB production, PhaR no longer binds to the granules and transcribes the phasing gene (phaP), slowing total PHB production. This explains why being in the log phase, when phasins are being created, causes the bacteria to synthesize great amounts of PHB. Entry into the stationary phase, however, retards PHB production when phasins are no longer being transcribed, and the bacteria’s rapid utilization of the polymer that remains eventually eliminates the presence of the polymer in the bacterial cells completely. Therefore, the bacteria must be harvested just when it is entering the stationary phase in order to collect the most polymer from the cells. Fig. 11