IN SCHOOL ARTICLE
Determination of the Optimal Conditions for
Polyhydroxybutyrate Accumulation in R. rubrum
Zoey Sloate1*, Susan Johnson2, Nicolas Guerin2, Carol Sullivan2, and Karin Spahl3
Student1, Teacher2: Wachusett Regional High School: 1401 Main Street, Holden, MA 01520
Advisor3: Regional Science Resource Center Laboratory, University of Massachusetts Medical School:
222 Maple Avenue, Shrewsbury MA 01545
*Corresponding author: zoslo21@gmail.com
Abstract
Synthetic plastics are ubiquitous. They are used for
everything from bottles to bags to buildings, and are
being produced in such excessive quantities that their
environmental consequences are staggering. The resin
that constitutes a large percentage of finished synthetic
plastic products is made from petroleum, and is virtually
resistant to decomposition, leading to environmental
damage, increased dependence on fossil fuels, industrial
pollution, safety hazards, and litter. However, an attractive
alternative could be provided by photosynthetic bacteria,
such as Rhodospirillum rubrum, which can produce
the plastic polymer polyhydroxybutyrate (PHB). This
bacteria is capable of producing biodegradable polymers
in an economically feasible way: by using inexpensive
substrates like acetic acid vinegar, being incubated in
dark conditions, and being exposed to nitrogen. PHB is
environmentally safe after it is discarded; this material can
be decomposed by anaerobic microorganisms, decreasing
dependence on fossil fuels and synthetic polymers. Yet
despite these advantages, synthetic plastics remain to
be dominantly used in commercial applications because
of their low cost. If biodegradable plastic production
could become produced more efficiently, it could replace
synthetic plastics and their detrimental effects. R. rubrum
was incubated on three different medias that were selected
based upon their potential for PHB accumulation in
cultures of Rhodospirillum rubrum. These three medias
were Luria Broth (LB) agar, 3% acetic acid with LB agar,
and 3% acetic acid. This bacteria, prior to being incubated
on these three medias, was incubated for 24 hours in the
presence or absence of light. It was predicted that the
culture that was first grown in the absence of light and
then on the acetic acid/LB agar media would produce the
most PHB plastic. A 3% acetic acid media incubated in the
absence of light produced the most PHB granules in the R.
rubrum cells.
Introduction
The fabrication and use of synthetic plastics has had profound
affects. The staggering quantity of the material clogs landfills,
leaches chemicals in to the environment, and poses a health hazard
to wildlife. Synthetic plastics’ resin is created from petroleum,
therefore increasing dependence on fossil fuel consumption.1
Yet despite these detrimental effects of synthetic polymers, it is
unlikely that society will stop using plastics, and return to using
metal, stone, glass, or wood for tasks that plastics now occupy.
Plastics are so popular because of their ideal qualities: they are
waterproof, stain repellant, stretchable, lightweight, durable,
malleable, and perhaps most importantly, cheap.2 Consumer
restraint can’t be expected to curb the use of synthetic plastics,
and neither can recycling. Every time a plastic is recycled, it is
heated and remolded for a new product. When this occurs, the
polymers that the plastic consists of are sheared, making the new
finished plastic product weaker than what it was before. This
makes the new product only suitable for a task less arduous than
the one it previously had. Thus, as a plastic is recycled, it becomes
weaker and weaker, until the plastic’s polymers have been sheared
and weakened to the point that it is not suitable for any viable
commercial use; at which point the plastic would be discarded into
a landfill. Synthetic polymers are recalcitrant, and take thousands
of years to decompose; a process that is delayed for even longer
in a landfill, due to the lack of air circulation and sunlight.1
A solution to the synthetic plastics problem is replacing them
with biodegradable plastics, defined as a degradable plastic in
which degradation results from microorganisms, such as bacteria
or fungi.1 A biodegradable plastic’s resin can be decomposed
much more quickly than that of a synthetic plastic, and certain
biodegradable plastics, such as polyhydroxybutyrate (PHB), can
be decomposed by anaerobic bacteria. For PHB to decompose,
it must be away from air, such as buried in a landfill. This turns
out to be an important benefit, because while the lack of air in a
landfill prevents synthetic plastics from degrading, this lack of air
facilitates the decomposition of PHB plastic. This biodegradable
plastic can take advantage of the current waste system. Since
its decomposition relies upon an anaerobic environment, the
consumer does not have to be concerned with his or her PHB
product decomposing while it is being used.3
In addition to decomposing biodegradable plastics, bacteria
can also produce them. The polyesters bacteria can produce
are called polyhydroxyalkanoates (PHAs), the most common
type being polyhydroxybutyrate (PHB). The polyesters occur as
granules inside the cells, and serve as an energy storage for the
bacterium.1 There are three main conditions under which bacteria
produce this material. However, the common link between them
is that the cell must first undergo growth and development,
and then be exposed to conditions that provoke polymer
accumulation. The first such condition is when photosynthetic
bacteria, such as Rhodobacter sphaeroides or Rhodospirillum
rubrum, undergo fermentation.4
In cellular respiration, during glycolysis, glucose is converted
to pyruvate, which is then sent to the mitochondria of the cell,
and converted to acetate. Coenzyme A then bonds with the
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Zoey Sloate, Susan Johnson, Nicolas Guerin, Carol Sullivan, and Karin Spahl
Page 2 of 5
acetate, forming acetyl-CoA. That enzyme is responsible for carrying the molecule to the next part of the cellular respiration reactions,
the Krebs cycle, but it cannot continue without oxygen.5 Therefore, the acetyl-CoA monomer is stopped and saved by being synthesized
and undergoing polymerization into a PHB polymer; a process that does not require adenosine triphosphate (ATP).6 For a photosynthetic
bacterium, this anaerobic cellular respiration occurs in the absence of light, since it produces food and grows in the light, and resorts to
its food supply in the absence of dark.5
The second such condition in which PHAs can be produced by bacteria is when there is acetic acid, or acetate, introduced to the cell.7
The cell can uptake the acetate, and can convert it to acetyl-CoA. Because of the abundance of acetate, an excess of acetyl-CoA is created,
which can then be stored and synthesized into PHB in the same fashion as anaerobic cellular respiration.8
A third reason that causes biodegradable plastic accumulation in bacteria is when there is an increase of nitrogen introduced to the cell.
Although the precise reason is unknown, it has been observed that an increase in nitrogen in a photosynthetic bacterium’s media leads to
an increase in PHB production. The speculated explanation for this occurrence is that since PHB is an readily oxidizable source of carbon,
it can provide electrons to power the nitrogen fixation reactions of the cell. Thus, it is believed that the bacterium creates more PHB to
compensate for the surplus of nitrogen that it must excrete.9
One specific bacterium that is receptive to all of the aforementioned conditions is the photosynthetic bacterium, Rhodospirillum
rubrum. This bacteria is classified with a biosafety level 1, the safest possible category for bacteria.10 This bacterium is also cataloged as
non-pathogenic by the United States.11 Photosynthetic bacteria is organized into groups based on various characteristics they have: if they
are oxygenic or anoxygenic, use hydrogen sulfide or organic compounds as an electron donor, and if chlorophyll is the main light-absorbing
pigment, or if rhodopsin is. Rhodospirillum rubrum is a purple non-sulfur bacterium, meaning it is anoxygenic, and uses organic electron
donors and rhodopsin.6 This bacteria can also be grown in aerobic conditions when not exposed to light, and can have carbon monoxide
be its only energy source to survive. This bacterium is traditionally incubated at 25˚ Celsius.12 Luria Broth (LB) agar is the standard media
that this bacteria is grown on. LB agar is a special type of agar that provides proteins, vitamins, and trace elements– such as nitrogen, in
the form of tryptone– that are essential for bacterial growth.13
There were two factors studied: light and type of media. For light, there were two conditions studied: incubation in the light for
24 hours and incubation in the absence of light for 24 hours. In light, the bacterium’s normal conditions allow the cell to undergo
photosynthesis, preventing cellular respiration and the creation of acetyl-CoA. Once the bacteria is transferred onto mediums of LB agar,
LB agar with 3% acetic acid, and 3% acetic acid, the presence of the additional acetic acid could create conditions more optimal for PHB
production. The surplus of acetic acid, a low molecular weight carbon substrate for the bacterium, could create a surplus of acetyl-CoA,
and then be synthesized into PHB. However, nutrients are necessary for the growth of bacteria, and thus the LB agar with acetic acid
will provide the proper reactants to sustain cell growth, while also providing the acetic acid. With the LB agar, there will be no acetic acid
available to be used in PHB synthesis, but the presence of nitrogen from the tryptone will help promote this molecule to form because of
its role in nitrogen fixation. However, this alone would not create excessive amounts of PHB, and would need acetic acid to do so. Likewise,
a media of only acetic acid will not provide enough amino acids, vitamins, and trace elements for the cell to function and properly convert
the acetic acid to acetyl-CoA to PHB at its full potential.
Based upon the aforementioned research, it was predicted that if cultures of Rhodospirillum rubrum are first grown in light and dark,
and then transferred to separate medias of Luria Broth (LB) agar, 3% acetic acid with LB agar, and 3% acetic acid, then the culture that is
first grown in the absence of light and then on the acetic acid/LB agar media will produce the most PHB granules.
Materials and Methods
The presence of acetic acid and nitrogen were tested on the bacterium Rhodospirillum rubrum to observe how they affected PHB
accumulation in the cell. However, it is important to note that in this experiment, the bacteria was only tested for when it was in the
presence of nitrogen, rather than being exposed to a surplus. In order to test the nitrogen and acetic acid variables, the bacteria plates
with different medias were prepared. For each test, two plates of each media were made, and there were three different medias used. A
Luria Broth (LB) agar plate was made, by boiling and dissolving 3 grams of LB agar into 300 mL of water, to attain 1% concentration of
LB agar. A portion of the solution was then poured into two 65 x 15 mm sterilized plastic bacteria plates, autoclaved, and allowed to set.
The second plate, the acetic acid plate, was prepared by dissolving 3 grams of pure agar into 296.6 mL of water. 3.3 mL of 3% acetic acid
vinegar was then added to the solution, and then a portion was poured into 2 bacteria plates, with the same dimensions as the plates listed
above. These plates were autoclaved, allowed to cool, and set. The third variable plate, the LB agar with acetic acid plate, was prepared by
dissolving dissolving 3 grams of LB agar into 296.6 mL of water, with 3.3 mL of 3% acetic acid vinegar then added to the solution, and
then a portion poured into 2 like bacteria plates. The LB agar plate provided the nitrogen variable for the cell; the acetic acid plate, which
had no additional nutrients, provided acetic acid; and the acetic acid/LB agar plate provided both the nitrogen and the acetic acid. The
bacteria used was a photosynthetic bacterium, Rhodospirillum rubrum, and was incubated at 25°C for 24 hours, either in the presence or
absence of light. Under these conditions, the bacteria was incubated on a 65 x 15 mm plate, on LB agar. The bacteria plates were sealed
by wrapping strips of Parafilm M Barrier Film around the edge, with each strip approximately 10 cm by 2 cm. This was done to create an
anaerobic environment for the bacteria. Initially, tube and liquid cultures were used rather than a plate culture, but it was determined that
both did not allow for enough bacteria growth to be adequately tested. One bacteria plate was incubated in each light condition, and the
bacteria grown on each plate after 24 hours was then spread onto the three variable plates as described above. This created a total of six
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Figure 1. Flow Chart of Growth Conditions of R. rubrum and the Transfer of Variables.
variable combinations. To elaborate, the bacteria on that been incubated in the light for 24 hours was spread from its initial plate to the
three variable plates: the acetic acid, LB agar, and the acetic acid with LB agar plates; and the same was done for the plat that had initially
been incubated in the dark. The growth conditions Rhodospirillum rubrum underwent in this experiment and the transfer of variables are
illustrated in Figure 1. The variable combination of light incubation, and the substrate of LB agar was the control of the experiment, as
those are the standard growing conditions for Rhodospirillum rubrum. After the bacteria had been spread onto the variable plates, they too
were wrapped with 10 cm by 2 cm strips of Parafilm M Barrier Film, to create an anaerobic environment for the bacteria. These six plates
were then incubated at 25°C for 48 hours in the presence of light. In order to test the quantity of PHB that had formed in the bacteria
cells after the three days of incubation, microscope smears were prepared. Microscope slides were prepared by using an eppendorf pipette
to measure 5 microliters of water onto the center of a 2.5 cm by 7.6 cm microscope slide. Then, an inoculation ring, after being sterilized
using a flame and allowed to cool, was used to spread a colony of bacteria from the plate to the microscope slide.. There were a total of 10
slides per plate, and in the creation of those slides, all of the bacteria from the plate was distributed between all of the slides. The bacteria
was then emulsified by the inoculation ring into the water droplet, making a smear in the center of the slide. The slide was then left to air
dry, so the excess water could evaporate and the bacteria could be secured to the slide. The slide was then passed slowly through a flame 3-4
times to ensure the bacteria was fixed. Next, in order to see the PHB granules that had formed, the microscope slide was stained with two
dyes: Sudan Black B and safranin O. The Sudan Black B stain, a lipid stain, was prepared as a .3% weight/volume solution in 60% ethanol,
and the safranin O stain was prepared as a .5% weight/volume in aqueous solution. The microscope slide was stained for 10 minutes with
the Sudan Black B solution, and if at any point the stain began to dry, more was added, to ensure that the stain was fully in contact with the
fixed bacteria the entire time. Safranin O was then administered immediately after the Sudan Black B, for 30 seconds. The dyes were then
gently rinsed off the slide with distilled water and carefully patted dry. The Sudan Black B dye stained the intracellular PHB granules a dark
blue or black color, and the safranin O made the cell’s cytoplasm appear pink or clear, which provided contrast from the granules. It was
assumed that the Sudan Black B dye only stained the PHB polymers. 10 microscope slides were made from each of the six variable plates,
for each trial. The slides were labeled either “a,” “an,” or “agar,” for the acetic plate, the acetic acid/LB agar plate, and the LB agar plate, to
mark which slide held bacteria from which variable plate. The slides were also labeled with a dash or a “d” in the upper right hand corner,
to mark whether the bacteria on that slide had been incubated in light or dark conditions, and a number, between 1 and 10. After the slides
were smeared, the PHB granules were distinct and definable when viewed under a light microscope. Each slide was viewed under a light
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Zoey Sloate, Susan Johnson, Nicolas Guerin, Carol Sullivan, and Karin Spahl
Page 4 of 5
microscope at 1000x magnification, and the PHB granules were counted. It is important to note that the entire smear was not observed,
only one view of the smear. To be consistent for each slide, there was a mechanism on the stage of the microscope that held every slide
in the same orientation, ensuring that the same area, for every slide, was viewed. However, this could have impacted the results, as not all
of the PHB granules were counted per slide. To count the PHB granules, tallies were drawn on paper. Every time the viewer came across
a blue-black dot, or a PHB polymer, inside a bacteria cell, a tally was recorded. The tallies were then counted on the paper to produce a
specific number of PHB granules that could be associated with that particular slide; or one data point. This was done for all the microscope
slides. All the steps listed above were then repeated to execute a second trial.
Results
It is important to note that all data points were a result of a viewer
counting PHB granules when viewed under a microscope, and as a
result, every statistic, or n, has a possibility of human error; which
is ±√n. The first method for data analysis used was basic addition
of the PHB granules for each of the six variable combinations for
the two trials conducted, revealing that the acetic acid/ LB agar
media under light conditions produced the most PHB, with a total of
6,633 granules. Following that was the acetic/dark combination, with
5,044, agar/light at 4,600, agar/dark with 4,306, acetic/light at 3,750,
and acetic acid LB agar at 3,493. When arranged in order of PHB Table 1. Total PHB Granules per Treatment.
levels, as is done in Table 1, and illustrated in Figure 2, there is no
discernible trend or pattern of variables and light conditions on the
PHB outcome; neither with the type of media nor the light condition.
Despite the irregularity presented here, further analysis illuminated
which combination to be the best for PHB accumulation.
Two-tailed, unpaired, unequal variance Student t-tests were then
used to compare each of the two trials together for like media variables,
to see if levels of PHB recorded for one trial were consistent to the
second trial. For example, acetic acid’s first light trial was compared
to acetic acid’s second light trial. All of the variables involving dark
conditions were found to be inconsistent between trials, proven by a
p value of .0001 for the agar and acetic medias, and .001 for the acetic
+ LB agar medium. Much lower than the accepted p value of .05 for
biology, these values rendered the dark trials as unreliable and invalid; Figure 2. Total PHB Granules per Treatment.
disproving the null hypothesis. However, all the combinations that
had been exposed to light during the first 24 hours of testing had p
values higher than .05; LB agar had a p value of .34, acetic acid had
.058, and acetic acid/LB agar had .05. These p values suggest that the
null hypothesis is false; more PHB was not created by the dark trials,
as predicted. This narrowed the viable variable combinations to only
those that had been initially exposed to light.
The light trials’ data points, for the LB agar, acetic acid/LB agar,
and acetic acid medias, were then applied to a box and whisker plot,
to compare the individual amounts of PHB counted per microscope
slide to like data points of the other medias, shown in Figure 2. This
was done to ensure that the acetic acid/LB agar’s superior PHB total,
as shown in Table 1, was not due to high outliers. From the plot,
shown in Figure 3, the acetic acid/LB agar maximum of 584, and
minimum of 170 demonstrated a higher range than that of acetic acid
(364, 58) and LB agar (460, 58). This again showed the acetic acid/LB
agar media in the light to be the best variable combination for PHB Figure 3. Box and Whisker Plot of Light Trials’ Totals.
accumulation.
Another set of two-tailed, unpaired, unequal variance t-tests were done to compare both light and dark trials of like medias to one
another, to see if the light conditions had a significant difference on the PHB production of the cell. For the acetic acid and LB agar variables,
when the 4 trials (two light and two dark trials) were compared to one another, their p values were greater than .05. This confirmed the null
hypothesis that the light and dark trials were the same, showing that those in the light conditions did not have a significant difference on
the PHB accumulation for those medias. This was done to the acetic acid/LB agar tests, but here, its p value was .0001, rejecting the null
hypothesis and proving that there was a difference in PHB accumulation between the light and dark variables.
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Zoey Sloate, Susan Johnson, Nicolas Guerin, Carol Sullivan, and Karin Spahl
Discussion
When the PHB granules for each variable combination were
totaled, it showed that the bacteria grown in the acetic acid/
LB agar media under light conditions produced the most PHB.
However, when the variable combinations were organized in order
of their PHB totals, there was no pattern regarding the type of
media or the light conditions; neither variable produced uniform
effects. Initial t-tests showed that the dark trials themselves
were not consistent to one another, and the second t-tests that
compared the light and dark trials showed that only the acetic
acid + LB agar media was affected by the light conditions. The
light trials’ minimums and maximums, when depicted onto a box
and whisker plot, revealed higher levels of PHB accumulation in
the acetic acid /LB agar media than other medias. This analysis
shows that not only did the LB agar/ acetic acid media in the light
produce the most PHB plastic, but was also the most reliable.
Only this combination was consistent between trials, and was the
only one to have the expected outcome: to be effected by the light
conditions.
The data obtained from this experiment did not support
the hypothesis; the acetic acid/LB agar media in the dark
combination that the hypothesis predicted actually produced the
least amount of PHB when compared to the the other variables,
whereas that same media in the light did the best. The hypothesis
for this experiment was proposed after considering the expected
behavior of Rhodospirillum rubrum in light and dark conditions,
and adding them to the predicted effects brought on by changing
the variables in the media. But rather, each test produced results
entirely because of the independent circumstances resulting
from the combination of the variables. The unpredicted results
could have been due to unknown confounding variables. Due
to the irregular nature of the results, more testing and research
would be required to better understand the relationship of PHB
accumulation in Rhodospirillum rubrum.
Further extensions of this project would be to rearrange
the order in which the bacteria was introduced to the variables;
for example, expose to the variable medias first, and then to the
dark or light conditions. Despite abundant research found about
the dark’s influence on PHB production, it was found that for
two of the three medias, it produced no significant difference.
Also, another area that could be extended upon in this project
is the amount of time the bacteria is left to incubate when
exposed to the variables, which would possibly promote more
PHB accumulation. A better way to have measured the PHB
would have been either through gas chromatography or by using
hemocytometer coverslip onto the microscope slides. The PHB
was counted with the human eye in this experiment, and thus the
statistics were very prone to human error. Also, technology that
could evenly spread the smear of bacteria onto the bacteria slide
would be optimal for future expereiemtns; as would viewing the
entire bacteria smear on the microscope slide for PHB.
Page 5 of 5
2. Grossman, Elizabeth. Chasing Molecules: Poisonous Products,
Human Health, and the Promise of Green Chemistry.
Washington DC: Island/ Shearwater, 2009.
3. Schwartz, Mark. “Biodegradable Plastic - Sustainable Built
Environment - Woods Institute.” Woods Institute for the
Environment. Web. 20 Feb. 2011. <http://woods.stanford.edu>
.
4. “Biological Macromolecules.” UNM Biology News. Web. 28
Nov. 2010. <http://biology.unm.edu>.
5. Danks, Susan M., E. Hilary Evans, and Peter A. Whittaker.
Photosynthetic Systems: Structure, Function, and Assembly.
Chichester Sussex: Wiley, 1983.
6. “Photosynthetic Bacteria.” University of Maryland College of
Chemical and Life Sciences. Web. 11 Aug. 2010. <http://www.
life.umd.edu>.
7. Zaborsky, Oskar R. Biohydrogen. New York: Plenum, 1998.
8. Sockett, R.E. “Methylation-Independent and MethylationDependent Chemotaxis in Rhodobacter
Sphaeroides
and
Rhodospirillum Rubrum.” Web. 14 Nov. 2010. <http://www.
ncbi.nih.gov>.
9. Wang, H. “Metabolic Regulation of Nitrogen Fixation in
Rhodospirillum Rubrum” Biochemical Society Transactions 34
(2006).
10. National Renewable Energy Laboratory. Photobiological
Production of Hydrogen. Golden, Colorado: U.S. Department
of Energy.
11. “Non-Pathogenic Bacteria.” Bacteria. Web. 22 Sept. 2010.
<http://www.phac-aspc.gc.ca>.
12. “DLC-ME | The Microbe Zoo” DLC-ME Home Page. Web.
4 Oct. 2010. <http://microbezoo.commtechlab.msu.edu>.
13. Bertani, Giuseppe. “Lysogeny at Mid-Twentieth Century:
P1, P2, and other Expereimental Systems -- Bertani 186 (3):
595.” The Journal of Bacteriology. American Society for Microbiology, Feb. 2004. .
References
1. Stevens, E. Green Plastics: an Introduction to the New Science
of Biodegradable Plastics. Princeton: Princeton UP, 2002.
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