FIG. 1: TEM image of PHA granules inside Ralstonia eutropha

1
The Production of Bioplastics Using the Waste
Stream of an Anaerobic Digester Elisabeth
Linton, Dr. Sridhar Viamajala, and Dr. Ronald C.
Sims Department of Biological and Irrigation
Engineering, Utah State University
RESULTS
3. PHA Production using the Waste Stream
The results of the anaerobic/aerobic cycling
experiment with the agricultural waste are given
as Figure 11. The aqueous PO4 concentration
decreases aerobically and increases
anaerobically. This indicates that PAOs are
naturally present in the waste. Also, the
fluctuation in PO4 concentration becomes more
pronounced with each cycle, which indicates PAO
growth and proliferation.
There are two major problems with conventional
plastics 1) They are derived from nonrenewable
fossil fuels, which are decreasing in
1. Calibration of Nuclear Magnetic Resonance
An obtained NMR spectrum for PHB is given as
Figure 4. Each of the labeled peaks correspond to
the hydrogen-bonds at carbon positions1, 2, and 3
in the PHB structure. Larger images
supply and increasing in cost, and 2. They are
not biodegradable. These issues have escalated
the need for alternatives. Polyhydroxyalkanoates
(PHAs) are a class of microbially accumulated,
biodegradable plastics.
of these peaks are shown as Figures 5-7.
The widespread use of PHAs is limited by cost.
PHAs are roughly 2.5 times more expensive than
traditional plastics. In an effort to improve the
economic outlook of bioplastics, this project is
focused on using anaerobically digested dairy
waste for PHA production. This eliminates the
cost of the carbon source, which typically
accounts for about one-third of the total
expense. Essentially, the
CH2 (3)
FIG. 5 Multiplet peak at 5.2 ppm
FIG. 6 Doublet at 1.2 ppm
FIG. 1 TEM image of PHA granules inside
Ralstonia eutropha
FIG. 11 Orthophosphate levels in waste during
anaerobic/aerobic cycling.
concept of this project is to use waste to
produce high-value bioplastics.
Samples were analyzed using the NMR method to
determine PHA content. The peaks obtained
correspond to those of the bioplastic standard.
These peaks, as shown in Figures 12 and 13,
confirm the presence of
FIG. 4 NMR spectrum for PHB standard.
FIG. 7 Multiplet peak at 2.5 ppm
Calibration for PHB was carried out using
specific concentrations of a PHB standard with
the NMR method. The area of each peak is
proportional to PHB content. Figure 8 depicts
this linear relationship.
CH (2)
CH2 (3)
FIG. 12 Multiplet at 5.2 ppm
FIG. 13 Multiplet at 2.5 ppm
FIG. 8 Plot of peak area versus known
concentration of PHB standard.
METHODS
bioplastics in the waste. The bioplastic
concentration was determined using the NMR
calibration curve as 0.18 mg PHA/ml of waste.
1. PHA Detection Methods
A Jeol ECX-300 spectrometer (Fig. 2) was used for
300 MHz 1H NMR. The position and the splitting
patterns of these signals relate to the
compound structure. Fluorimetry was also used to
detect PHAs and reinforce the results of the NMR
method.
2. Pure Culture Growth Studies
A growth curve was constructed for A. vinelandii
by determining protein fluctuations (Fig. 9).
Samples (7mL) were periodically collected and 10
mg of dry cells were analyzed using the NMR
method. The max product yield was determined as
3.74 mg PHB/10 mg of cells. Fluctuations in
bioplastic content were also determined using
fluorimetry (Fig. 10).
2. Pure Culture Growth Studies
Azotobacter vinelandii was grown on a sucrose
medium. This organism produces polyhydroxybutyrate
(PHB), which is the most prevalent type of PHA.
FIG. 2 NMR spectrometer
3. Phosphorus-Accumulating Organisms (PAOs)
PAOs are microorganisms associated with
bioplastic accumulation. Aerobically, PAOs uptake
orthophosphate (PO4) and degrade PHAs. In the
absence of oxygen, they release PO4 and produce
PHAs. The presence of these organisms in the
waste was determined by cycling between anaerobic
and aerobic states and monitoring the PO4
concentration.

• Utah Science and Technology Research Initiative
  (USTAR) Biofuels Initiative
• USU Sustainable Energy Research Center
• Utah State University Undergraduate Research
  Office

FIG. 3 Cycling Agricultural Waste
FIG. 10 Qualitative and quantitative bioplastic
content in A. vinelandii
FIG. 9 A. vinelandii growth curve