Transcript Production of Poly Hydroxybutyric Acid with B megaterium

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 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.
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
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.
Fig. 4
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
PHB (μg/mL)
Absorbance (AU)
@ 235 nm
0
0
2
2
0.2338
1.8
4
0.4676
1.6
6
0.7014
8
0.9352
10
1.169
12
1.403
14
1.637
16
1.871
PHB Spectrophotometric Assay
Absorbance (%) at 235 nm
1.4
1.2
Standard
1
0.8
0.6
Standard for 160μg Poly-β-Hydroxybutyric Acid
0.4
2
0.2
0
2
4
6
8
10
12
14
16
1.5
Poly-β-Hydroxybutyric Acid μg/mL
Fig. 5
1
Absorbance (%)
0
Detection of Poly-β-Hydroxybutyric Acid in Carbon Sources
3
Standard
0.5
2.5
789
770
751
732
713
694
675
656
637
618
599
580
561
542
523
504
485
466
447
428
409
390
371
352
333
314
295
276
257
238
219
200
0
Absorbance (%)
2
Fig. 6
-0.5
Control
Glucose
Glycerol
Maple Syrup
1.5
1
λ Wavelength (nm)
Table 2 Amount of PHB Present in Each Media
0.5
Carbon Source
Absorbance (AU)
@ 235 nm
PHB (μg/mL)
Control
0.078
0.67
Glucose
0.96
8.2
Glycerol
0.336
2.87
Maple Syrup
2.315
19.80
20
0
20
7
21
4
22
1
22
8
23
5
24
2
24
9
25
6
26
3
27
0
27
7
28
4
29
1
29
8
30
5
31
2
31
9
32
6
33
3
34
0
34
7
35
4
36
1
36
8
37
5
38
2
38
9
39
6
0
Fig. 7
λ Wavelength (nm)
Bacteria Growth Over Time Measured by Absorbance
Fig. 8
Table 3 Bacteria Growth in Media of Various
Carbon Sources
1.8
1.6
1.4
Absorbance (%)
1.2
1
26 hours
71 hours
0.8
26 hours
71 hours
0.6
26 hours
71 hours
0.4
26 hours
71 hours
0.2
0
25 Control
26 Control
27 Control
28 29 30 31 32 33 Glucose Glucose Glucose Glycerol Glycerol Glycerol
Number of Bacteria Sample and Type of Media
34 Maple
Syrup
35 Maple
Syrup
36 Maple
Syrup
Test Tube Number
Absorbance (AU)
@ 235 nm, Day 2
Absorbance (AU)
@ 235 nm, Day 4
25
0.743
1.237
26
0.732
1.290
27
0.732
1.272
28
0.988
1.062
29
1.037
1.028
30
1.064
1.048
31
0.753
0.819
32
0.794
0.889
33
0.820
0.916
34
0.524
1.559
35
0.544
1.586
36
0.548
1.449
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.
Bacterial Growth Over Time Measured by Absorbance
1.8
1.6
Bacterial Projected Growth Phases
Control
Stationary
Glucose
Glycerol
Maple Syrup
Log
Death
Lag
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.
Absorbance at 420nm (%)
1.4
1.2
1
26 hours
71 hours
0.8
26 hours
71 hours
0.6
26 hours
71 hours
0.4
26 hours
71 hours
0.2
0
Control
Glucose
Glycerol
Number of Bacteria Sample and Type of Media
Maple Syrup
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 surfaceto-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.
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. 11