Bacterial Plastics
AOS-HCI Project Proposal
Alexander Lim Geng Wang
Sim Meng Ying
Nicholas Kee Jia Hao
Introduction
Rationale
Plastics are currently widely utilized. About 75 billion pounds of plastics are
produced a year for industrial purposes. Plastics serve as a very important material in
everyday life, due to its durability and ability to take different kinds of shapes. Thus it
can be used to manufacture containers, stationery and lab apparatus.
Although plastics are very useful, they are xenobiotic, thus explaining its
resistance to microbial degradation. Since it is non-biodegradable, disposal methods
of plastics would include incineration and landfills. However, the process of
incineration releases harmful gases such as hydrogen chloride and hydrogen
cyanide. Furthermore, incineration is not cost effective. On the other hand, landfills
are reaching their maximum capacity and the problem of land scarcity restricts the
number of landfills that are available. As such, there have been rising occurrences of
plastics being discarded into the marine environment. Consequently, marine life is
being compromised.
Bacteria belonging to the genera Clostridium, Syntrophomonas,
Pseudomonas, and Alcaligenes are able to synthesize the polymer PHA poly(3-
hydroxyalkanoate) when subjected to nutrient-poor environments, osmotic pressure,
and ultraviolet radiation. Copolymer of PHA, PHB (poly-B-hydroxybutyrate), is known
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to have similar characteristics as polypropylene, which is used to make medical
instruments and clothing.
Objectives
1) To determine the carbon source that results in the highest growth rate of
Alcaligenes eutrophus
2) To determine the conditions of growth (carbon and nitrogen source, duration
of growth) which can yield the highest amount of polymer in A. eutrophus
3) To find out the best method of lysis for the cell pellets after growth in
fermentation medium
3 current different lysis methods
1) Sodium Hypochlorite
2) Sodium Dodecyl Sulphate (SDS) + Sodium Hypochlorite
3) Lysozyme + Sodium Dodecyl Sulphate (SDS) + Sodium
Hypochlorite
4) To determine the most economically viable carbon source that can result in
the highest growth of A. eutrophus.
Hypothesis
1) The bacterium A. eutrophus shows different growth rates when grown in
different carbon sources such as glucose, fructose and lactose.
2) Different polymers are produced by A. eutrophus when grown in different
carbon sources.
3) The lysis method using lysozyme yields the highest amount of polymer.
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Literature Review
Research has been done by other researchers in this field of study. It has
been found out that many types of bacteria can synthesize PHA and PHB (Fiechter,
1990), such as Alcaligenes eutrophus and Pseudomonas. Hence, researchers
started to analyse the different factors and results in the production of polymers.
Some researchers have done some studies on the effects of the synthesized polymer.
Ayorinde, Saeed, Price, Morrow, Collins, McInnis, Pollack, Eribo carried out a
research on the effect of saponified vernonia oil on the properties of synthesized
PHB. Hahn, Chang and Lee had also tried to characterize the properties of PHB
produced by Alcaligenes eutrophus and Escherichia coli, such as the melting and
boiling point, and tensile strength. Other than characteristics of the polymers,
researchers have also researched on varying the substrate fed to the bacteria
Alcaligenes eutrophus and Pseudomonas (Kocer, Borcakli, Demirel, 2002).
Results obtained so far:
Alcaligenes eutrophus was grown in glucose, lactose and fructose and the following
results were obtained.
The cells were lysed using different methods and the following results were obtained.
Results for lysis and bacterial growth
Carbon NaClO / g Lysozyme / g
source/Method Cell Polymer Cell Polymer Cell Polymer
Weight Mass % Weight Mass % Weight Mass %
Fructose 0.14 0.0324 23.1 0.1078 0.0019 1.76 0.0685 0.0013 1.9
Glucose NA NA NA - - - 0.0764 0.002 2.62
Yield of polymer from A. eutrophus was slightly higher when grown in glucose than
fructose
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Summary of findings:
A.eutrophus grew at the highest rate in fructose, followed by glucose.
It showed a low growth rate in lactose.
New methods
a) Food Waste (fruit peels)
1) Terpenes
2) Phenolic compounds
3) Pectins
Possibility of using fruit peel extracts as carbon sources.
b) Potential of using waste products from sugarcane stalk, bagasse
which can be used broken down into simpler forms to manufacture
biodegradable food packaging.
c) Seeds
1) Starch
2) Proteins
3) Fats
Possibility of activating enzymes present in the cotyledon to break
down the macromolecules into simpler soluble substances. Seed
extracts can then be prepared during germination phase to obtain
sugars etc. The extracts can then be used as carbon sources.
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New lysis methods
1) Sonicator
2) Qiagen column lysis method
Experimental Procedure
Maintaining the stock culture of the bacterium
Trypticase soy agar was prepared with a concentration of 40 g/l. It was then
autoclaved at 121°C for 15 minutes and poured into Petri dishes. The agar is then
left to solidify and dried. The stock culture of A. eutrophus was streaked onto the
agar plate and incubated at 26°C overnight. Subculturing of bacterial cells was done
once every 2 weeks.
Growth of bacterium
A. eutrophus was precultured in 10ml of trypticase soy broth (TSB). It was incubated
at 26°C for 24 h at 120 rpm. The next day, the absorbance of preculture was taken at
600 nm with a spectrophotometer and the absorbance was standardized between 1.2
and 1.5.
5 ml of the overnight preculture was inoculated into the main culture consisting of 100
ml of TSB and grown for about 22 h at 26°C at 120 rpm. The overnight culture in TSB
was centrifuged at 4000 rpm for 10 min to obtain the cell pellet. The cell pellet was
then washed once with sterile water and resuspended in a small volume of
fermentation medium. The resuspended cell pellet was transferred to 300 ml of
fermentation medium and grown for 4-7 d at 26°C in a shaker at 120 rpm.
Composition of fermentation medium:
Fermentation medium (Kim et al., 1995)
o 3.8g Na2HPO4, 2.7g KH2PO4, 2.0g NH4Cl, 0.2g MgSO4, 1ml
trace minerals solution per litre
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o Carbon source (20 g/l) (fructose, lactose or glucose)
o Trace minerals: FeCl3 9.7, CaCl2 7.8, CuSO4 0.156, CoCl2 0.119,
NiCl2 0.118, CrCl2 0.062 g/l
Harvesting of cells
Cells were harvested by centrifugation at 4000 rpm for 10 min. The resulting cell
pellet was washed twice with sterile water, resuspending fully in it each time. The cell
pellet was then transferred to Petri dishes and dried at 55°C overnight. The dry
weight of cells was recorded.
Recovery of polymer using sodium hypochlorite
This procedure used sodium hypochlorite to cause lysis of cells, thus releasing the
polymer (Hahn et al., 1995). The dried cell pellet was resuspended in 10% sodium
hypochlorite solution (1.8% wt / vol) and left overnight at 30°C in a waterbath. The
polymer was separated from the aqueous fraction containing cell debris by
centrifugation at 8000 rpm for 10 min and the polymer was collected as a pellet. It
was washed with sterile water and transferred to a Petri dish to be dried at 55C
overnight, weighed and finally stored at 4°C.
Recovery of polymer using sodium dodecyl sulphate – sodium hypochlorite treatment
20 ml of 1% sodium dodecyl sulphate solution was added to the dried cell pellet and
incubated at 55C for 15 min, after which 20 ml of 10% sodium hypochlorite solution
was added and incubated at 30C overnight. The following day, the mixture was
centrifuged and the polymer was obtained as a pellet. It was washed with sterile
water and dried overnight at 55C before weighing.
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Recovery of polymer using lysozyme – sodium dodecyl sulphate – sodium
hypochlorite treatment
The cell pellet was resuspended in 10 ml of SET buffer containing 5 mg/ml lysozyme
and incubated at 30°C for 30 min. The suspension was centrifuged at 8000 rpm for
10 min and the cell pellet was washed with sterile water. 10 ml of 0.2 M sodium
hydroxide solution / 1% sodium dodecyl sulphate were added to the cell pellet and
30°C for 45 min. Following centrifugation and washing with sterile water, 10 ml of
10% sodium hypochlorite solution were then added and incubated at 30°C for 45 min.
The suspension was centrifuged, washed with sterile water, dried at 55°C and
reweighed.
Proposed lysis method for Qiagen column
1) Add sterile water to the cell pellet in the proportion of 50mg of cells to 180µl.
2) Add proteinase K and Buffer AL (lysis buffer) in the proportion of 50mg of cells to
20µl proteinase K and 200µl Buffer AL. Incubate at 56C
Polymer Characterization
Melting point: Melting Point Apparatus
Molecular Mass: Mass Spectroscopy/ Gel Permeation Chromatography
Chemical Structure: Nuclear Magnetic Resonance
Possible instruments:
HPLC, GC
X-ray Diffraction, X-ray Crystallography, SEM
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References
Ayorinde, F.O., Saeed, K.A., Price, E., Morrow, A., Collins, W.E., McInnis, F., Pollack,
S.K. and Eribo, B.E. (1998). Production of poly-(-hydroxybutyrate) from saponified
Vernonia galamensis oil by Alcaligenes eutrophus. Journal of Industrial Microbiology
and Biotechnology 21: 46 – 50.
Hahn, S.K., Chang, Y.K. and Lee, S.Y. (1995). Recovery and characterization of
poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant
Escherichia coli. Applied and Environmental Microbiology 61: 34 – 39.
Kim, H.-Y., Park, J.-S., Shin, H.-D. and Lee, Y.-H. (1995). Isolation of glucose
utilizing mutant of Alcaligenes eutrophus, its substrate selectivity, and accumulation
of poly--hydroxybutyrate. Journal of Microbiology 33: 51 – 58.
Madison, L.L and Huisman, G.W. (1999). Metabolic engineering of poly(3-
hydroxyalkanoates): from DNA to plastic. Microbiology and Molecular Biology
Reviews 63: 21–53.
Paustian, T. (1998). Bacterial plastics. Retrieved February 16, 2008 from
http://www.bact.wisc.edu/Microtextbook/index.php?name=Sections&req=viewarticle&
artid=155&page=1
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