This document summarizes research on recovering polyhydroxyalkanoates (PHA) bioplastics produced by Ralstonia eutropha H16 bacteria using aqueous two-phase systems (ATPS). The ATPS used co-polymers of ethylene oxide and propylene oxide (EOPO) with various molecular weights and salts to separate PHA. Phase diagrams were constructed to identify optimal EOPO-salt compositions. PHA was found to accumulate primarily in the top phase when using an ATPS of 44.6% EOPO3900 and ammonium sulphate at a volume ratio of 1, achieving a purification factor of 67%. Further experimentation is needed to improve results reliability.

1. Yi Theng Wong, Pau Loke Show*
School of Chemical and Environmental Engineering, Faculty of Engineering ,
University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia
Bio-plastic production in an aqueous two-phase green system
Dean’s Undergraduate Research Scheme (DURS) 2014
Abstract
The research is the study of the recovery of polyhydroxyalkanoates
(PHA) from Ralstonia eutropha H16 using aqueous two-phase system
(ATPS) composed of co-polymer of EOPO and salt.
0%
10%
20%
30%
40%
50%
60%
0% 5% 10% 15% 20%EOPO970(wt/wt)
(NH4)2SO4 (wt/wt)
(a)
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10%
20%
30%
40%
50%
60%
0% 10% 20% 30% 40%
EOPO2500(wt/wt)
(NH4)2SO4 (wt/wt)
(b)
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10%
20%
30%
40%
50%
60%
0% 10% 20% 30%
EOPO3900(wt/wt)
(NH4)2SO4 (wt/wt)
(c)
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5%
10%
15%
20%
25%
30%
35%
0% 10% 20% 30% 40%
EOPO3900(wt/wt)
Na2HPO4 (wt/wt)
(d)
Figure 3(a-d) Phase diagram of EOPO copolymer and salt. (a-c) at room temperature,
(d) at 40ºc
 To obtain phase diagram using co-polymer with molecular weight of
970,2500 and 3900 and salts
 To separate PHA produced using aqueous two-phase system
 To identified the optimum separation by varying Mw of co-polymer,
type of salt, tie-lie length and volume ratio
Scope of Research
Research Findings
Conclusion & Future work
1. Kaul, A., The Phase Diagram, in Aqueous Two-Phase Systems: Methods
and Protocols, R. Hatti-Kaul, Editor. 2000, Humana Press. p. 11-21.
2. Show, P.L., et al., Primary recovery of lipase derived from Burkholderia
cenocepacia strain ST8 and recycling of phase components in an aqueous
two-phase system. Biochemical Engineering Journal, 2012. 60(0): p. 74-
80.
References
Methodology
Introduction
 Offers simple, easily scalable,
energy efficient and mild
separation technique for bio-
separation
 Temperature-induced
separating polymer - EOPO
 Has a low cloud point -
recyclable
 High plastic usage level leads to plastic pollution.
 Replace conventional plastic with bio-plastic
 Comes out with a economical way of separating bio-plastic produced
Acknowledgement
The author appreciately acknowledge the technical support from supervisor Dr
Show Pau Loke, phd student Yoong Kit, Leong and assistance of Zi Bin, Tan in
constructing phase diagram of EOPO/disodium hydrogen phosphate.
 Plastic which is made from
biomass or biodegradable
 PHA studied in the
experiment are obtained
from Ralstonia Eutropha
H16 through fermentation
of glucose
Bio-based
Bio-
degradablePHA
Figure 1 Recycle scheme of EOPO [2]
The optimum condition for recovery of PHA is achieved using ATPS
composed of EOPO3900 and ammonium sulphate with 44.6% TLL, volume
ratio of 1 which gives a purification factor of 67%. The experiment has to
be repeated to improve the reliability of the result.
 Disodium hydrogen phosphate can’t dissolve in water under normal room temperature which
increases the complexity in constructing phase diagram
 The time taken to form two-phase is decrease as molecular weight of co-polymer increases
Cell + PHA
Table 1 Partitioning of PHA in EOPO3900 and ammonium sulphate
 PHA mostly accumulates at the top phase of ATPS
TLL VR PF
41.10 1.00 0%
44.00 2.33 22%
44.60 1.00 67%
46.70 1.90 10%