J Polym Res (2017) 24:189

DOI 10.1007/s10965-017-1345-x

ORIGINAL PAPER

Synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)
with high 4HB composition and PHA content using 1,4-butanediol
and 1,6-hexanediol for medical application
Hambali Norhafini 1,2 & L. Thinagaran 3 & K. Shantini 3 & Kai-Hee Huong 1,3 &
Ishak Muhammad Syafiq 1 & Kesaven Bhubalan 1,4 & A. A. Amirul 1,3,5

Received: 22 May 2017 / Accepted: 28 September 2017

# Springer Science+Business Media B.V. 2017

Abstract Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) 2.1-fold 4HB monomer composition higher compared to the

[P(3HB-co-4HB)] with high 4HB monomer harbors enhanced
biocompatibility and mechanical properties, which are useful
as implantable and absorbable biomaterial in medical and
pharmaceutical fields. Transformant Cupriavidus sp.
USMAA1020 with an additional PHA synthase gene, phaC
was found to produce P(3HB-co-4HB) with 86 mol% of 4HB
monomer composition and high PHA content of 69 wt% in
shake flask cultivation using mixed substrate of 1,6-
hexanediol and 1,4-butanediol. Single-stage cultivation in
3 L fermentation has confirmed the ability of this strain to
produce high 4HB monomer composition of 95 mol% in large
scale fermentation with 75 wt% PHA content and high PHA
concentration of 18.7 g/L. Interestingly, this strain was capa-
ble of surviving higher carbon concentration (1.05 wt% C)
than the wild-type strain (0.69 wt% C). The present study
results in P(3HB-co-4HB) with 1.3 and 2.3-fold PHA content
and concentration respectively, with the ability to accumulate
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10965-017-1345-x) contains supplementary
material, which is available to authorized users.
* A. A. Amirul
amirul@usm.my
1
Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM,
11700 Gelugor, Penang, Malaysia
2
Chemical Engineering Faculty, Universiti Teknologi Mara,
40450 Shah Alam, Selangor, Malaysia
3
School of Biological Sciences, Universiti Sains Malaysia, 11800
Minden, Penang, Malaysia
4
School of Marine Science and Environment, Universiti Malaysia
Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia
5
Centre for Chemical Biology, Universiti Sains Malaysia, 11900
Bayan Lepas, Penang, Malaysia
wild-type strain. A higher specific growth rate of 0.123 h−1
accompanied by high product yield, Yp/x of 3.8 times more
than the wild-type strain were also obtained. Image from TEM
showed cells with PHA content of 75 wt%, whichwere occu-
pied with significant PHA granule. This copolymer possesses
an average molecular weight (Mw) and a polydispersity index
of 156 kDa and 3.5 respectively with a tensile strength, elon-
gation at break and Young’s modulus of 22.9 MPa, 463.2%
and 187.3 MPa respectively. This polymer has a glass transi-
tion temperature (T g) and melting temperature (T m ) of
−48.9 °C and 61.9 °C respectively.
Keywords P(3HB-co-4HB) . 4HB monomer . Mixed
substrate . PHA synthase . Transformant Cupriavidus sp.
USMAA1020
Introduction
Polyhydroxyalkanoate (PHA) has potential to substitute syn-
thetic polymer as PHA films can be fully degraded in soil
within 50 days [1]. It is currently being developed as absorb-
able biomaterial for medical and pharmaceutical applications.
PHA-based biomaterials possess a new variety of properties
that broaden the design of existing biomaterials, hence
allowing the growth of new and improved products [2, 3]. A
wide range of bacteria is capable of producing PHA as intra-
cellular energy storage, when the surrounding environment is
excess of carbon source but lacks of growth nutrients, such as
nitrogen, phosphorus or micro-elements [4]. Poly(3-
hydroxybutyrate-co-4-hydroxybutyrate) P(3HB-co-4HB) is
one of the scl-PHAs, known to be produced by Cupriavidus
necator (formerly known as Ralstonia eutropha). Other
P(3HB-co-4HB producing bacteria include Hydrogenophaga
189 Page 2 of 9
pseudoflava [5, 6], Comamonas acidovorans [7, 8] and
Alcaligenes latus [9, 10].
In medical applications, biocompatibility is one of the
most important properties in which the biomaterial does
not trigger negative biological response in vivo during
degradation in a living organism. Both 3HB and 4HB
are natural metabolites found in human blood and natural-
ly distributed in the mammalian body, therefore P(3HB-co-
4HB) has good biocompatibility and is well tolerated
in vivo [11]. In addition, its biodegradability and biocom-
patibility properties make it possible to be implanted in
the human body without having to be surgically removed
thereafter [12]. In fact, P(4HB) was the first biopolymer
approved for clinical applications as an absorbable suture
(TephaFLEX) by Food and Drug Administration (FDA)
[13]. High 4HB monomer in P(3HB-co-4HB) contributes
to rapid degradation that varies with porosity, thus suitable
for tissue regeneration applications where gradual loss of
implant mass and steady growth of new tissue are required
[12]. In addition, an increase in the surface wettability of
P(3HB-co-4HB) nano-fiber construct, incorporated with
collagen peptide, was observed with an increase in 4HB
monomer from P(3HB-co-20 mol%4HB) to P(3HB-
co-82 mol%4HB). The increased surface wettability sup-
ports cell growth and its application as biodegradable
wound dressing [14].
Several attempts had been done by researchers to in-
crease the 4HB monomer composition in P(3HB-co-4HB),
which included cultivation by using sole or mixed carbon
sources, adding of additives/precursors, using recombinant
strains and production by single and two-stage cultivation
methods [15–18]. As a result, the present study was aimed
to study the production of P(3HB-co-4HB) with high 4HB
monomer composition using transformant Cupriavidus sp.
USMAA1020 with additional phaC gene. The strain was
cultivated using a mixture of 4HB precursors at varying
concentration in single-stage cultivation. Although mixed-
substrate strategy is well established for the enhancement
of PHA production, its effect on the production of P(3HB-
co-4HB) with high 4HB monomer composition using
strains with additional phaC gene has not been extensively
studied.
Materials and methods
Bacterial strain and plasmid maintenance
The transformant Cupriavidus sp. USMAA1020 used in this
study harbors an additional copy of PHA synthase gene, as
reported previously [19]. The transformant Cupriavidus sp.
USMAA1020 was streaked on nutrient agar (NA) and incu-
bated for 24 to 48 h at 30 °C. Kanamycin solution (50 μg/mL)
J Polym Res (2017) 24:189
was added into nutrient agar for plasmid maintenance and
stability. For long term storage, the bacterial strain was kept
in glycerol stock 40% (v/v) containing kanamycin (50 μg/mL)
at −20 °C [19].
Biosynthesis of P(3HB-co-4HB)
Growth and P(3HB-co-4HB) accumulation in shake flask
fermentation
Inocula (0.1 g/L) were transferred into 50 mL mineral
medium in 250 mL Erlenmeyer flasks containing 50 μg/
mL kanamycin solution, 3.70 g/L KH2PO4, 5.80 g/L
K2HPO4, 1.1 g/L (NH4)2SO4, 0.2 g/L MgSO4·7H2O and
1.0 mL/L trace elements solution (2.78 g FeSO4·7H2O,
1.98 g MnCl2·4H2O, 2.81 g CoSO4·7H2O, 1.67 g CaCl2·
2H2O, 0.17 g CuCl2·2H2O and 0.29 ZnSO4·7H2O per L
of 0.1 mol/L HCl) [20]. The medium was supplemented
with the mixture of two, out of four types of carbon
sources (1,6 hexanediol, 1,4-butanediol, γ-butyrolactone
and sodium 4-hydroxybutyrate), which were sterilized sep-
arately by autoclaving or by membrane filtration
(0.20 μm). The fermentation was carried out for 48 h.
Each set of experiment was carried out in triplicates.
Batch production of P(3HB-co-4HB) in bioreactor
Inocula (0.1 g/L) were transferred into a 3 L mineral medium
in benchtop fermenter (Sartorius Biostat B plus, Germany)
containing 50 μg/mL kanamycin solution. This culture medi-
um was supplemented with both carbon sources (1,6-
hexanediol and 1,4-butanediol) at a ratio of 1:5 and total con-
centration of 0.75 wt% C. The fermentation was carried out at
30 °C with an agitation speed and aeration rate of 300 rpm and
1 vvm respectively. The optical density of biomass was mon-
itored every 12 h at a wavelength of 540 nm. The experiment
was carried out in triplicates.
Analytical determination
Lyophilized cell materials were analyzed for their PHA con-
tent, 3HB and 4HB monomer compositions using gas chro-
matography equipped with SBP-1 column (Shimadzu GC-
2014, Japan). Approximately 15–20 mg of lyophilized cell
was subjected to methanolysis in the presence of 2 mL chlo-
roform and 2 mL methanol sulphuric acid [85%:15% (v/v)].
The mixture was then heated at 100 °C for 2 h and 20 min.
Once cooled, 1 mL of distilled water was added and vortexed.
The organic layer containing the reaction products was ana-
lyzed using gas chromatography (GC) according to the stan-
dard method [20].
The molecular weight of the polymer was analyzed with
gel permeation chromatography (GPC) using a Shimadzu LC-
J Polym Res (2017) 24:189
9A system equipped with a refractive index detector (RID-
10A) and a PLgel mixed C column (Polymer Laboratories,
Ltd., UK) at 40 °C. Chloroform was used as the eluent at a
flow rate of 1.0 mL/min with sample concentrations and in-
jection volumes of 1.0 mg/mL and 10 μL respectively.
Polystyrene standards with low polydispersity were used to
construct a calibration curve and the molecular weight was
calculated using Mark-Houwink equation. The Mark-
Houwink constant for polystyrene in chloroform was taken
as K = 0.011 mL/mg and α = 0.73, whereas K = 0.016 mL/
mg and α = 0.76 were taken for P(3HB) in chloroform [21].
The thermal data for the polymer were determined using
differential scanning calorimeter (Pyris DSC, Perkin-Elmer,
US). The pure polymer of approximately 8.0–10.0 mg was
encapsulated in aluminum pans and then heated from
−50 °C to 200 °C at a heating rate and cooling rate of 10 °C
min−1 and 20 °C min−1 respectively. The melting temperature
(Tm), glass transition temperature (Tg) and enthalpy of fusion
(ΔHm) were taken based on the heat transmission profile [22].
The tensile strength, Young’s modulus and elongation at
break of the polymers were determined using a tensile ma-
chine (GOTECH A1–3000 with U60 software, Taiwan) with
load cell capacity of 100 N. A standard test piece (75 mm
length × 4 mm width) was cut in a dumbbell shape and
gripped at either end by suitable apparatus in the tensile ma-
chine which slowly exerted an axial pull at the rate of 10 mm
min−1 until it was broken. The average value of eight repli-
cates was taken for each sample. The tensile tests were ana-
lyzed according to ASTM D638–93 [23].
Results
Effects of mixed substrate on bacterial growth, PHA
accumulation and 4HB monomer
Four structurally related 4HB substrates (1,4-butanediol, 1,6-
hexanediol, γ-butyrolactone and sodium 4-hydroxybutyrate)
were mixed at varying concentrations. Effects on the 4HB
monomer composition and productivity of P(3HB-co-4HB)
using transformant Cupriavidus sp. USMAA1020 were stud-
ied. According to Babel and co-workers, the combination of
more than one physiologically similar substrate provides a
simultaneous utilization, which can overcome the limitation
faced by single-stage cultivation in product biosynthesis
hence increasing the production yield [24]. This had been
demonstrated by Huong and co-workers through their work
when the usage of mixed-substrate strategy increased the PHA
concentration and content compared to using sole carbons [15,
25]. This study employed the same approach to evaluate the
feasibility of mixed-substrate culture strategy on the
transformant strain that has an additional copy of PHA syn-
thase gene.
Page 3 of 9 189
Results showed a notable increase in PHA accumulation
when the transformant strain was cultured in mixed-substrate
(refer Table 1). For instance, both γ-butyrolactone and sodium
4-hydroxybutyrate (4HB-Na) accumulated only 48 wt% and
30 wt% PHA content respectively during sole carbon study.
However, when both carbons were combined with high ratio
of 1,4-butanediol, the PHA accumulated up to 71 wt% (γ + 1,4)
and 79 wt% (4HB-Na + 1,4). The same trend was also observed
when 1,6-hexanediol was combined with high ratio of 1,4-
butanediol whereby 82 wt% of PHA content was recorded com-
pared to 61 wt% obtained during sole carbon study. This incre-
ment can only be seen when carbons were mixed with 1,4-
butanediol. Among all the substrate mixture, 1,6-hexanediol
and 1,4-butanediol with 1:5 ratio produced the highest 4HB
monomer composition of 93 mol% with PHA content and
PHA concentration of 82 wt% and 7.60 g/L respectively.
Effect of different total carbon concentration on bacterial
growth, PHA accumulation and 4HB monomer
Prior to fermenter studies, the optimum total carbon concen-
tration of 1,6-hexanediol +1,4-butanediol (1:5) was investigat-
ed. The total carbon concentrations was tested in the range of
0.3 wt% C to 1.35 wt% C. Based on Table 2, carbon concen-
tration of 0.75 wt% C (C/N = 32) was optimum as it accumu-
lated the highest PHA content, PHA concentration and 4HB
monomer at 76 wt%, 9.7 g/L and 84 mol% respectively.
Interestingly, the transformant strain was capable of with-
standing high concentration of carbon as no significant reduc-
tion was observed in PHA content, PHA concentration and
cell residual when a maximum of 1.05 wt% C (C/N = 45) of
total carbon were used.
Biosynthesis of P(3HB-co-4HB) in 3 L bioreactor
The mixed-substrate of 1,6-hexanediol +1,4-butanediol
(1:5) and the overall total carbon concentration of
0.75 wt% C were used in 3 L fermentation. At a cultiva-
tion period of 108 h, the PHA has occupied 75 wt% of
the total biomass, which contributed to the highest PHA
concentration of 18.7 g/L. Besides, the 4HB monomer was
also found to increase from 70 mol% at 24th hour to a
maximum of 95 mol% at the 108th hour (refer Fig. 1). As
for the comparison studies, the wild-type strain
Cupriavidus sp. USMAA1020 was cultured with the same
carbon mixture and same total carbon concentration in 3 L
fermentation. Results have shown that there was only
59 wt% and 8.7 g/L of PHA content and PHA concen-
tration being accumulated respectively, with a 4HB mono-
mer composition of 46 mol% (refer to Table 3). This has
led to a conclusion that the transformant strain showed
1.3- and 2.3-fold higher PHA content and PHA concen-
tration respectively, with its undeniable ability to
 189 Page 4 of 9 J Polym Res (2017) 24:189

Table 1 Effect of mixed-substrate on copolymer P(3HB-co-4HB) production

Carbon combination C1:C2 (wt% C) CDW (g/L) PHA content (wt%)f PHA concentration (g/L)f Residual biomass (g/L)g PHA composition
(mol %)

3HBf 4HBf

1,6 + 1,4 0.1 0.5 9.3 ± 0.7ab 82 ± 6a 7.6 ± 0.9ab 1.7 ± 0.5a 7 ± 0c 93 ± 0a
0.2 0.4 10.0 ± 0.3a 82 ± 4a 8.2 ± 0.3a 1.8 ± 0.4a 10 ± 0c 90 ± 0ab
0.3 0.3 8.4 ± 0.3ab 78 ± 3a 6.6 ± 0.4ab 1.9 ± 0.3a 19 ± 0ab 81 ± 0bc
0.4 0.2 8.9 ± 0.0b 73 ± 5a 6.0 ± 0.9b 2.2 ± 0.5a 24 ± 1a 76 ± 1c
0.5 0.1 5.0 ± 0.3c 69 ± 5a 3.4 ± 0.0c 1.6 ± 0.0a 15 ± 1bc 85 ± 1ab
1,6 + γ 0.1 0.5 2.3 ± 0.1a 11 ± 3b 0.2 ± 0.1c 2.0 ± 0.0a 38 ± 6a 62 ± 6b
0.2 0.4 2.2 ± 0.2a 14 ± 1b 0.3 ± 0.1c 2.0 ± 0.1ab 23 ± 10b 77 ± 10a
0.3 0.3 2.4 ± 0.2a 28 ± 2c 0.7 ± 0.0b 1.7 ± 0.2b 16 ± 2b 83 ± 2a
0.4 0.2 2.0 ± 0.1a 38 ± 1b 0.8 ± 0.0b 1.3 ± 0.1c 13 ± 2b 87 ± 2a
0.5 0.1 2.4 ± 0.2a 55 ± 3a 1.3 ± 0.1a 1.1 ± 0.1c 10 ± 2b 90 ± 2a
1,6 + 4HB-Na 0.1 0.5 3.4 ± 0.0b 37 ± 7b 1.2 ± 0.2c 2.1 ± 0.2a 26 ± 2a 74 ± 2b
0.2 0.4 3.6 ± 0.3b 38 ± 1ab 1.4 ± 0.1c 2.3 ± 0.1a 26 ± 3a 74 ± 3b
0.3 0.3 4.4 ± 0.1a 48 ± 0ab 2.1 ± 0.0ab 2.3 ± 0.1a 21 ± 1a 79 ± 1b
0.4 0.2 4.4 ± 0.1a 49 ± 2a 2.2 ± 0.1a 2.2 ± 0.0a 11 ± 1b 89 ± 1a
0.5 0.1 3.9 ± 0.3ab 47 ± 6ab 1.8 ± 0.1b 2.0 ± 0.4a 11 ± 2b 89 ± 2a
1,4 + γ 0.1 0.5 5.9 ± 0.2d 49 ± 5b 2.9 ± 0.3c 3.0 ± 0.3a 69 ± 2e 31 ± 2e
0.2 0.4 6.2 ± 0.1cd 49 ± 2b 3.1 ± 0.1c 3.1 ± 0.2a 60 ± 4d 40 ± 4d
0.3 0.3 6.4 ± 0.2c 51 ± 3b 3.2 ± 0.2c 3.2 ± 0.2a 51 ± 0c 49 ± 0c
0.4 0.2 8.6 ± 0.0b 68 ± 0a 5.9 ± 0.0b 2.7 ± 0.0a 40 ± 3b 60 ± 3b
0.5 0.1 9.5 ± 0.2a 71 ± 4a 6.7 ± 0.5a 2.8 ± 0.3a 28 ± 4a 72 ± 4a
1,4 + 4HB-Na 0.1 0.5 5.5 ± 0.2b 49 ± 6b 2.7 ± 0.4d 2.8 ± 0.3a 21 ± 2a 79 ± 2b
0.2 0.4 5.6 ± 0.2b 53 ± 2b 3.0 ± 0.2cd 2.6 ± 0.1ab 21 ± 1a 79 ± 1b
0.3 0.3 6.3 ± 0.1b 56 ± 5b 3.5 ± 0.2c 2.8 ± 0.3a 18 ± 1ab 82 ± 1ab
0.4 0.2 8.3 ± 0.3a 69 ± 1a 5.7 ± 0.3b 2.5 ± 0.0ab 17 ± 4ab 83 ± 4ab
0.5 0.1 8.3 ± 0.1a 79 ± 2a 6.5 ± 0.3a 1.8 ± 0.2b 13 ± 1b 87 ± 1a
γ + 4HB-Na 0.1 0.5 2.7 ± 0.0c 31 ± 1b 0.9 ± 0.0b 1.9 ± 0.0c 31 ± 1 69 ± 1ab
0.2 0.4 3.3 ± 0.1b 34 ± 4b 1.1 ± 0.2b 2.2 ± 0.1ab 31 ± 1 69 ± 1ab
0.3 0.3 3.3 ± 0.1b 33 ± 1b 1.1 ± 0.1b 2.2 ± 0.1ab 29 ± 4 71 ± 4a
0.4 0.2 3.5 ± 0.1b 31 ± 6b 1.1 ± 0.2b 2.4 ± 0.2ab 38 ± 5 62 ± 5b
0.5 0.1 5.0 ± 0.4a 43 ± 0a 2.1 ± 0.2a 2.9 ± 0.2a 50 ± 3 50 ± 3c

a-e
Data show the mean ± standard deviation of three replicates. Means with different superscripts within the same column are significantly different at
P ≤ 0.05 level (Tukey test). 1,6: 1,6-hexanediol; 1,4: 1,4-butanediol; γ: γ-butyrolactone; 4HB-Na: Sodium 4-hydroxybutyrate
f
Data determined from GC analysis
g
Data determined by subtracting PHA concentration from cell dry weight

accumulate high 4HB monomer composition which was transformant strain possessed higher specific growth rate
2.1-fold higher compared to the wild-type strain. The of 0.123 h−1, and accompanied by high product yield,

Table 2 Effect of total carbon concentration in mixed-substrates on copolymer P(3HB-co-4HB) production

Total Carbon (wt% C) C/N CDW (g/L) PHA content (wt%)g PHA concentration (g/L)g Residual biomass (g/L)h PHA composition (mol %)

3HBg 4HBg

0.3 13 6.2 ± 0.4f 50 ± 1d 3.1 ± 0.2d 3.1 ± 0.2ab 28 ± 0bc 72 ± 0bc

0.45 19 9.6 ± 0.4cd 67 ± 1c 6.4 ± 0.2bc 3.2 ± 0.2ab 22 ± 2cd 78 ± 2ab
0.60 26 11.5 ± 0.2ab 68 ± 3cd 7.8 ± 0.3ab 3.7 ± 0.3a 25 ± 0cd 75 ± 0ab
0.75 32 12.8 ± 0.3a 76 ± 1ab 9.4 ± 0.7a 3.4 ± 0.4a 17 ± 3d 83 ± 3a
0.90 39 12.3 ± 0.4ab 74 ± 2abc 9.1 ± 0.4a 3.3 ± 0.2ab 16 ± 1d 84 ± 1a
1.05 45 11.7 ± 1.1bc 84 ± 0a 9.8 ± 0.9a 1.9 ± 0.2c 21 ± 4cd 79 ± 4ab
1.20 52 8.4 ± 0.8de 70 ± 2cd 5.9 ± 0.3bc 2.5 ± 0.2bc 37 ± 2ab 63 ± 2cd
1.35 58 7.5 ± 0.3ef 66 ± 1c 4.8 ± 0.3cd 2.6 ± 0.0bc 41 ± 5a 59 ± 5d
a-f
Data show the mean ± standard deviation of three replicates. Means with different superscripts within the same column are significantly different at
P ≤ 0.05 level (Tukey test)
g
Data determined from GC analysis
h
Data determined by subtracting PHA concentration from cell dry weight
J Polym Res (2017) 24:189 Page 5 of 9 189

Fig. 1 Biosynthesis of 100 25

Residual biomass and PHA

copolymer P(3HB-co-4HB) by 90

PHA content and 4HB

transformant Cupriavidus sp. 80 20

(wt% and mol%)

USMAA1020 in 3 L single-stage 70

concentration
composition
fermentation 60 15 PHA content

(g/L)
50
40 10 4HB monomer
30 PHA concentration
20 5 Residual biomass
10
0 0
0 12 24 36 48 60 72 84 96 108
Time (h)

Yp/x, of 3.8 times more than the wild-type strain. The
formation of PHA granule by the transformant strain was
observed using transmission electron micrograph as in
Fig. 2.
The physical and thermal properties of the copolymer pro-
duced from this transformant strain were shown in Table 4. The
average molecular weight (Mw) and the polydispersity index of
this polymer were 156 kDa and 3.5 respectively. On the other
hand, the tensile strength recorded was 23 MPa with the elon-
gation at break of 463%. The copolymer has a glass transition
temperature (Tg) and melting temperature (Tm) of −48.9 °C and
62 °C respectively.
Discussion
Among all the substrate mixtures, the combination of 1,6-
hexanediol and 1,4-butanediol with 1:5 ratio produced the
highest 4HB monomer composition, PHA content and PHA
concentration. The composition of the polymer synthesized is
governed primarily by three factors: the specificity of the PHA
synthase present, the carbon source, and the pathway by which
the supplied carbon source is metabolized. Based on Table 1,
highest 4HB monomer composition of 93 mol% was achieved
by transformant Cupriavidus sp. USMAA1020 which pos-
sessed an extra copy of the phaC gene. Lau and Sudesh had
mentioned that phaC gene may act as a bottleneck in P(3HB-
co-4HB) synthesis, which affects both the copolymer content
and the 4HB monomer composition [32]. In order to increase
4HB monomer composition, the gene dosage should be in-
creased. The same strategy was employed by Syafiq and co-
workers which resulted in a high 4HB monomer composition
of 93 mol% in shake-flask fermentation and a 4HB monomer
composition within a range of 83-89 mol% in large-scale fer-
mentation [19]. The increase in the phaC gene dosage also
improved PHA content. In the P(3HB-co-4HB) biosynthesis
pathway, 1,4-butanediol will be first oxidized to 4-
hydroxybutyric acid to form 4-hydroxylbutyryl-CoA, before
converted to 4-hydroxybutyrate, where only a part of it will
undergo a complex pathway to form 3-hydroxybutyrate (refer
Fig. 3) [23, 27]. Syafiq and co-workers found that, the
transformant Cupriavidus sp. USMAA1020 possessed higher
PHA synthase activity of 187 U/g compared to the wild-type
Cupriavidus sp. USMAA1020 (82 U/g) [19]. Generally, the
polymerization rate of the copolymer is catalyzed by the PHA
synthase gene present in the bacteria. Increasing the copy num-
ber of PHA synthase genes directly increases the PHA synthase
activity, which increases the polymerization rate of PHA [28].
The pathway of 4-hydroxybutyrate formation coupled with an
increase in the polymerization rate, due to the extra PHA syn-
thase gene, allowed an efficient utilization of 1,4-butanediol
towards PHA accumulation, therefore increasing PHA content.
The presence of 1,6-hexanediol in the combination seemed
to contribute to high PHA accumulation and 4HB monomer

Table 3 Comparison of growth

and production factors between Parameter Wild-type strain Transformant strain
wild-type Cupriavidus sp.
USMAA1020 and transformant Maximum specific growth rate, μmax (h−1) 0.115 0.123
Cupriavidus sp. USMAA1020 PHA production yield, Yp/x (gg−1) 1.2 4.4
during P(3HB-co-4HB) PHA production yield, Yp/s (gg−1) 0.6 1.5
biosynthesisa
Final PHA content, (wt%) 59 75
Final PHA concentration, (gL−1) 8.7 20.3
Final 4HB composition (mol %) 46 95
a
Inocula (0.1 g/L) was supplemented with both carbon sources (1,6-hexanediol and 1,4-butanediol) at ratio of 1:5
and total concentration of 0.75 wt% C. The fermentation was carried out in 3 L fermenter at 30 °C with agitation
speed and aeration rate at 300 rpm and 1 vvm respectively
189 Page 6 of 9
PHA granules
Fig. 2 Transmission electron micrograph image of P(3HB-co-
95 mol%4HB) granule with 75 wt% of PHA content produced from
transformant Cupriavidus sp. USMAA1020. Magnification: 20,000×,
Scale bar: 500 nm
composition. In the cell, 1,6-hexanediol is first oxidized to 6-
hydroxyhexanoyl-CoA and then converted into coenzyme A
thioester, which is converted into 4HB-CoA through ß-oxida-
tion and subsequently supplies 4HB monomer to the copoly-
mer chain [27]. Thus, the combination of 1,4-butanediol and
1,6-hexanediol substrates allows an efficient utilization
through the biosynthetic pathway towards both PHA biosyn-
thesis and cell growth, which consequently increased the cell
growth, PHA accumulation and 4HB monomer composition.
The cell growth, PHA accumulation and 4HB monomer com-
position using this substrate combination were found to be
even higher as compared to previous work by Syafiq and
co-workers, which utilized a substrate combination of 1,6-
hexanediol and γ-butyrolactone (high:low) on the same
transformant strain. Unlike the present study that used several
carbon mixtures, Syafiq and co-workers used one carbon mix-
ture (1,6-hexanediol and γ-butyrolactone) for the production
of P(3HB-co-4HB) with high 4HB monomer composition
[19]. The efficient utilization of 1,4-butanediol and 1,6-
hexanediol has also been demonstarted by Huong and co-
workers, which employed wild type Cupriavidus sp.
USMAA1020 [25]. This study has also resulted in higher
Table 4 Thermal and mechanical
properties of common PHA Properties Melting
polymers temperature,
Tm (°C)
Poly(3HB) 178
Poly(3HB-co-20%3 HV) 145
Poly(4HB) 53
Poly(3HB-co-95%4HB) 62
J Polym Res (2017) 24:189
PHA concentration with higher 4HB monomer composition
in shake flask through single-stage cultivation when compared
with previous studies [15, 29, 30] (refer Table 5).
This transformant strain is capable of surviving at
higher concentration of carbon compared to using the
wild-type strain, in which the cell growth and PHA accu-
mulation was inhibited at a total carbon concentration of
0.69 wt% [16]. It was reported that an introduction of an
extra gene in a genetically engineered cell enabled alter-
native functional enzymes and pathways, which posed as
advantageous backups to survive in a stress environment
[31]. Previously, a C/N ratio of 29 was reported as the
optimum ratio for cell growth and PHA accumulation
using the wild-type strain Cupriavidus sp. USMAA1020
[16]. Meanwhile, Mq. Iqbal & Amirul has shown that
C/N ratio of 30 was favorable for PHA production using
the wild-type strain Cupriavidus sp. USMAA2–4 [23].
Through the use of the wild-type strain of Cupriavidus sp.
USMAA1020, Amirul and co-workers had applied a broad
range of C/N ratio from 10 to 100 in single-stage cultivation,
where C/N 20 was the optimum ratio for PHA productivity
[20]. The optimum C/N ratio obtained in the present study
(C/N = 32) was higher than previously published data.
As shown in Fig. 2, bioreactor studies had produced a
PHA content of 75 wt%, whereby a large granule that
almost occupied the entire cell was observed. The high
PHA content of 75 wt% obtained in this study was higher
than the data reported by Syafiq and co-workers, whereby
64 wt% of PHA content was obtained using the same
strain in their bioreactor experiment with the mixture of
1,6-hexanediol and γ-butyrolactone [19]. The 4HB unit
composition increased from 70 mol% of 4HB for 24 h
of fermentation to 95 mol% at the end of the fermentation
period of 108 h. This is the highest so far in among all
the studies being performed in single-stage cultivation
using single-stage cultivation [16, 19, 32].
The data obtained from the present study proved that the
production of P(3HB-co-4HB) with high 4HB monomer com-
position can be enhanced when cultivated using the mixture of
Glass transition Tensile Elongation Reference
temperature, Tg strength at break (%)
(°C) (MPa)
4 43 5 [8]
-1 20 50 [26]
−48 104 1000 [8]
-49 23 463 This work
J Polym Res (2017) 24:189 Page 7 of 9 189

Fig. 3 Proposed biosynthesis pathway of P(3HB-co-4HB) copolymer using related carbon sources [23, 27]

1,4-butanediol and 1,6-hexanediol. Consequently, the use of
different carbon mixtures is feasible in improving the produc-
tion of P(3HB-co-4HB) with high 4HB monomer composi-
tion, which implies the significance of realizing the full poten-
tial of the mixed-substrate strategy.
The elongation at break possessed by this copolymer
(463.2%) was close to the previous study by Syafiq and co-
workers, where P(3HB-co-91 mol%4HB) was obtained using
the same strain with the elongation at break of 377% [19]. In
comparison to other polymers as in Table 4, both P(3HB) and
P(3HB-co-3 HV) have a high melting temperature of 178 °C
and 145 °C respectively making them difficult for fabrication
processes [8]. The copolymer obtained from this work pos-
sessed a lower melting point and glass transition temperature,
closely resemble to the thermal properties of P(4HB) (refer
Table 4). Martin and William mentioned that this range of
melting point and glass transition temperature allowed stable
melting process up to 200 °C and modest loss of molecular
mass, which broadens the thermal processing window of this
polymer [13]. While P(4HB) had been successfully applied in
tissue engineering to be used in implantable medical products,
a study by Grabow and co-workers mentioned that the native
leaflet valve, used in surgical heart valve disease, possessed
the tensile strength and elongation at break of 11 MPa and
23% respectively [33]. Interestingly, the tensile strength of
the polymer obtained from this study (22.9 MPa) is higher
compared to the value portrayed by the valve leaflet,
which possibly makes this polymer a good candidate to
be applied in tissue engineering scaffold for the fabrication
of leaflet valve.
Conclusion
This work confirmed that the transformant strain Cupriavidus
sp. USMAA1020, which possessed an extra PHA synthase
gene (phaC), has the ability to biosynthesize P(3HB-
co-4HB) with high 4HB monomer up to 95 mol%, as well
as with high PHA content and concentration when cultivated
using a mixed substrate of 1,4-butanediol and 1,6-hexanediol.

Table 5 P(3HB-co-4HB) production using bioreactor via batch fermentation

Microorganism Substrates PHA content PHA concentration 4HB unit References

(wt%) (g/L) composition (mol%)

Transformant Cupriavidus necator PHB¯4 Sodium 4-hydroxybutyrate 14 0.28 87 [32]

Cupriavidus sp. USMAA1020 1,4-butanediol +1,6-hexanediol 73 8.6 35 [16]
Transformant Cupriavidus sp. USMAA1020 1,6-hexanediol + γ-butyrolactone 64 5.6 89 [19]
Transformant Cupriavidus sp. USMAA1020 1,4-butanediol +1,6-hexanediol 75 20.3 95 This work
189 Page 8 of 9
Increments in PHA content, 4HB monomer composition, and
product yield, Yp/x, were observed using this strain compared
to using the wild-type strain. Interestingly, this study revealed
that the presence of an extra PHA synthase gene in the
transformant strain allows strain adaptation with higher car-
bon concentration compared to the wild-type strain. This poly-
mer also possesses better thermal and physical properties
when compared with other polymers, such as P(3HB) and
P(3HB-co-3 HV). In conclusion, this work has confirmed that
the mixed-substrate strategy used in the production of P(3HB-
co-4HB) with high 4HB monomer composition, using strain
with additional phaC gene, is a successful strategy in increas-
ing P(3HB-co-4HB) productivity.
Acknowledgements The authors acknowledge the research grants pro-
vided by the Ministry of Science, Technology and Innovation (02-05-23-
SF0023) and also the USM Research University Grant
(1001/PBIOLOGI/811304) that has resulted in this article.
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