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Polyhydroxyalkanoates (PHAs) are a family of biopolymers with good biodegradability. Poly(3- hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-25 mol % 4-hydroxybutyrate) [P(3HBco- 25 mol % 4HB)] and poly(3-hydroxybutyrate-co-75 mol % 4-hydroxybutyrate) [P(3HB-co-75 mol % 4HB)] were fabricated using the electrospinning technique to obtain fibers. Electrospun P(3HB) showed formation of fibers when 30 kV voltage was applied to 4 % P(3HB) extruded at 60 μL/min with prior heating for 15 min at 60 °C. Fabricated P(3HB-co-4HB) showed a continuous polymer mat with embedded beads formation. P(3HB) fabricated at different time of electrospinning (5, 10, 15 and 20 min) and concentrations (1 %, 2 %, 3 % and 4 %) subjected to in vitro enzymatic degradation by PHA depolymerase showed decrease in polymer weight. The highest rate of degradation was exhibited by 2 % P(3HB) electrospun for 15 min. Films of 1 % P(3HB-co-25 mol % 4HB) and 1 % P(3HBco- 75 mol % 4HB) subjected to in vitro lipase degradation also exhibited decrease in polymer weight. P(3HB-co-75 mol % 4HB) films showed significant decrease in weight compared to P(3HB-co-25 mol % 4HB). Degraded P(3HB) had fibril-like structures whereas P(3HB-co-4HB) surface structure became more porous. Environmental degradation of these polymers was successful with P(3HB-co- 4HB) being better degraded
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– 236 –
Journal of Siberian Federal University. Biology 2 (2015 8) 236-253
~ ~ ~
УДК 577.12
Fabrication and Degradation
of Electrospun Polyhydroxyalkanoate Film
Lee Joyyia,
Nanthini Sridewib, Amirul Al-Ashraf Abdullaha,
Ken-ichi Kasuyac and Kumar Sudesha*
aSchool of Biological Sciences
University Sains Malaysia
Penang, 11800, Malaysia
bNational Defence University of Malaysia
Kuala Lumpur, 57000, Malaysia
cDivision of Molecular Science
Gunma University
1-5-1 Tenjin, Kiryu, Gunma 376-8515, Japan
Received 19.01.2015, received in revised form 20.02.2015, accepted 16.05.2015
Polyhydroxyalkanoates (PHAs) are a family of biopolymers with good biodegradability. Poly(3-
hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-25 mol % 4-hydroxybutyrate) [P(3HB-
co-25 mol % 4HB)] and poly(3-hydroxybut yrate-co-75 mol % 4-hydroxybutyrate) [P(3HB-co-75 mol %
4HB)] were fabricated using the electrospinning technique to obtain bers. Electrospun P(3HB)
showed formation of bers when 30 kV voltage was applied to 4 % P(3HB) extruded at 60 μL/min
with prior heating for 15 min at 60 °C. Fabricated P(3HB-co-4HB) showed a continuous polymer mat
with embedded beads formation. P(3HB) fabricated at different time of electrospinning (5, 10, 15 and
20 min) and concentrations (1 %, 2 %, 3 % and 4 %) subjected to in vitro enzymatic degradation by
PHA depolymerase showed decrease in polymer weight. The highest rate of degradation was exhibited
by 2 % P(3HB) electrospun for 15 min. Films of 1 % P(3HB-co-25 mol % 4HB) and 1 % P(3HB-
co-75 mol % 4HB) subjected to in vitro lipase degradation also exhibited decrease in polymer weight.
P(3HB-co-75 mol % 4HB) lms showed signicant decrease in weight compared to P(3HB-co-25
mol % 4HB). Degraded P(3HB) had bril-like structures whereas P(3HB-co-4HB) surface structure
became more porous. Environmental degradation of these polymers was successful with P(3HB-co-
4HB) being better degraded.
Keywords: polyhydroxyalkanoates, biopolymers, electrospinning, in vitro, degradation.
DOI: 10.17516/1997-1389-2015-8-2-236-253.
© Siberian Federal University. All rights reserved
* Corresponding author E-mail add ress: ksudesh@usm.my
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
Изготовление и деградация пленок
из электроспряденного полигидроксиалканоата
Ли Джойиa, Нантини Сридевиб,
Амирул Аль-Ашраф Абдуллаa,
Кен-ичи Касуйяв, Кумар Судешa*
aШкола биологических наук
Университет науки Малайзии
11800, Пенанг, Малайзия
бНациональный университет обороны Малайзии
57000 Куала-Лумпур, Малайзия
вУниверситет Гуммы
1-5-1 Тенджин, Кирю, Гумма 376-8515, Япония
Полигидроксиалканоаты (ПГА) представляют собой семейство биополимеров c хорошей
биоразлагаемостью. С использованием электростатического формования были получены
пленки из волокон поли-3-гидроксибутирата [П(3ГБ)], поли-3-гидроксибутирата-co-4-
гидроксибутирата с содержанием 25 молярных % 4ГБ [П(3ГБ-co-25 мол. % 4ГБ)] и поли-3-
гидроксибутирата-co-4-гидроксибутирата с содержанием 75 мол. % 4-гидроксибутирата
[П(3ГБ-co-75 мол. % 4ГБ)]. Электростатическое формование П(3ГБ) после предварительного
нагрева в течение 15 минут при температуре 60 °C продемонстрировало формирование
волокон при воздействии напряжения 30 кВ на 4%-ный раствор П(3ГБ) при скорости подачи
раствора 60 мкл/мин. Полученный П(3ГБ-co- 4ГБ) представлял собой непрерывный полимерный
мат с вкрапленными гранулами. П(3ГБ), спряденный при различной продолжительности
электростатического формования (5, 10, 15 и 20 мин) и при разной концентрации растворов
(1, 2, 3 и 4 %), подвергнутый ферментативной деградации in vitro с использованием ПГА-
деполимеразы, показал уменьшение массы полимера. Наибольшая скорость деградации была
достигнута при электроформовании 2 % П(3ГБ) в течение 15 минут. У пленок из 1 % П(3ГБ-
co-25 мол. % 4ГБ) и 1 % П(3ГБ-co-75 мол. % 4ГБ), подвергнутых деградации липазой in vitro,
также было отмечено уменьшение массы полимера. Пленки из П(3ГБ-co-75 мол. % 4ГБ)
продемонстрировали значительное сокращение массы по сравнению с пленками из П(3ГБ-
co-25 мол. % 4ГБ). Деградированная полимерная пленка из П(3ГБ) имела фибриллярную
структуру, в то время как структура поверхности П(3ГБ-co-4ГБ) ста ла более пористой.
Данные полимеры успешно деградировали в окружающей среде, причем для П(3ГБ-co-4ГБ)
отмечалась более быстрая деградация.
Ключевые слова: полигидроксиалканоаты, биополимеры, электростатическое формование,
in vitro, деградация.
– 238 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
Introduction
Polyhydroxyalkanoates (PHAs) act as
intracellular storage compounds which provide
energy and carbon sources to enhance survival of
various environmental bacteria and archaea under
environmental stress. In vivo, they are amorphous
polymers which are synthesized in the presence
of excess carbon with limiting concentration of
at least one growth essential nutrient such as
nitrogen, phosphorus, sulfur or oxygen. PHAs
are accumulated intracellularly in the form of
granules. In laboratory settings, different carbon
sources can be fed to wild type or recombinant
strain microorganisms to produce different types
of PHA. PHA can be degraded by microorganisms
such as bacteria and fungi in various environment
such as soil, aquatic (sea water and lake water),
landlls and activated sludge. Bacteria degrade
PHA in the environment by secreting several
enzymes, such as PHA depolymerase, lipase and
esterase. In general, PHA accumulating bacteria
have intracellular depolymerase whereas some
non-PHA accumulating bacteria can produce
extracellular depolymerase to degrade PHA
derived from dead or lysed cells that accumulate
PHA (Jendrossek & Handrick, 2002). The
microorganisms excrete extracellular PHA
depolymerase to degrade environmental PHA and
utilize the decomposed compounds as nutrient.
Due to this biodegradable property, PHAs are
attractive as substitute for some non-degradable
petroleum based plastics.
Poly(3-hydroxybutyrate) [P(3HB)] was
discovered by Maurice Lemoigne in the 1920s
(Lemoigne, 1926). P(3HB) is stiff, crystalline and
brittle. This polymer consists of only one type
of monomer, 3-hydroxybutyrate (3HB). P(3HB)
can undergo three different kinds of degradation
which mainly are enzymatic degradation,
hydrolytic degradation and thermal degradation
(Zhao & Cheng, 2006). Degradation of P(3HB)
homopolymer by PHA depolymerase is a kind of
enzymatic degradation which can be intracellular
or extracellular (Mergaert et al., 2000).
The incorporation of monomer
4-hydroxybutyrate (4HB) into P(3HB) could
decrease the crystallinity and melting point of the
homopolymer. Thus, it improves the brittleness
and crystallinity of P(3HB). 4HB can be found
in the brain, heart, liver, lung, kidney and muscle
of mammalian body and degradable by lipases
in vivo by surface erosion process (Nelson et al.,
1981). P(3HB-co-4HB) copolymer can be degrade
by lipase produced from mammalian body and
is more suitable in tissue engineering (Grifth,
2002).
Electrospinning technique can be used to
fabricate polymer bers in the average diameter
range of 100 nm to 5 μm. These fabricated mats are
excellent scaffold that mimic the characteristics
of extracellular matrix as they provide large
surface area to volume ratios, exibility in
surface functionalities and high porosity (Ren et
al., 2008). Polymer bers are produced when the
electrical forces at the surface of polymer solution
in the spinneret overcome the surface tension and
cause an electrically charged jet to be ejected on
the surface of the electrically conductive collector
(Frenot & Chronakis, 2003).
Electrospinning technique had been
employed in this study in which the different
processing parameters and conditions were used
to produce PHA lms. The electrospun lms of
P(3HB), P(3HB-co-25 mol % 4HB) and P(3HB-
co-75 mol % 4HB) were characterized based on
their surface morphology via Scanning Electron
Microscopy (SEM) and degradation rate of
these lms were investigated under laboratory
conditions and in the environment.
Materials and methods
Materials
Poly(3-hydroxybutyrate) [P(3HB)], poly(3-
hydroxybuty rate-co-25 mol % 4-hydroxybutyrate)
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Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
[P(3HB-co-25 mol % 4HB)] and poly(3-
hydroxybuty rate-co-75 mol % 4-hydroxybutyrate)
[P(3HB-co-75 mol % 4HB)] were used in this
study. Chloroform was purchased from Fischer
Scientic, Malaysia. PHA depolymerase puried
from Ralstonia pickettii T1 (Kasuya et al., 1996)
was used and porcine lipase was purchased from
Sigma, Malaysia.
Preparation of polymer solution
and phosphate buffer solution
P(3HB), [P(3HB-co-25 mol % 4HB] and
[P(3HB-co-75 mol % 4HB] were dissolved in
chloroform at room temperature and were stirred
for two days before ltering with Sartorius
PTFE membrane lter (0.2 μm pore size).
Precursor solution of P(3HB) was rst prepared
at concentration of 1 %, 2 %, 3 % and 4 %
without prior heating before electrospinning to
investigate the optimum concentration. P(3HB)
polymer solution (4 %) and P(3HB-co-4HB)
polymer solution (1 %) were heated for 5, 10,
15 and 20 min at 60 °C and for 15 and 20 min
at 50 °C respectively before fabrication by
electrospinning. Disodium hydrogen phosphate
(Na2HPO4) was added into 100 mL of distilled
water to achieve the target molarity of 0.1 M. The
pH of 0.1 M Na2HPO4 was adjusted to 7.4 using
0.1 M HCl solution.
Electrospinning
Electrospinning was performed using
Esprayer™ ES-2000 (Fuence, Co. Ltd., Japan).
The polymer solution was loaded into a 1 mL
stainless steel glass syringe with an inner
needle diameter of 0.5 mm. The syringe was
set vertically at a distance of 20 cm from the
collecting plate. P(3HB) polymer solution of
different concentrations were fabricated at 15
kV and extrusion rate of 40 μL/min. Different
voltages and solution extrusion rates were applied
for other sample sets. The electrospun bers were
collected on the ber collection area at different
time intervals of 5, 10, 15 and 20 min.
In vitro degradation of electrospun lms
by PHA depolymerase and lipase
From the stock solution of 1.3 mg/mL,
6 μL of PHA depolymerase was added to 2
mL of phosphate buffer for the preparation of
depolymerase enzyme solution. An amount of
0.167 g of porcine lipase enzyme powder (30 U/
mg) was dissolved in 100 mL of distilled water
for preparation of 50 U/mL of stock solution.
Approximately 2 mL of this solution was used
for degradation of the electrospun lm samples.
Each lm was cut (1 cm × 1 cm), weighed and
incubated in depolymerase enzyme solution for
P(3HB) samples while P(3HB-co-25 mol % 4HB)
and P(3HB-co-75 mol % 4HB) were incubated in
lipase enzyme solution. The samples were placed
in an incubator at 37 °C for 30, 60, 90 and 120
min. The lms were removed from the enzyme
solution and washed gently with distilled water
before air-drying for one day. The weight of
lms was recorded and plotted in a line graph
as a function of incubation time (min). Polymer
weight loss was calculated as:
Initial weight (mg) –
Weight of lm after incubation (mg)
The rate of degradation was calculated as
follow:
[Initial weight of lm –
Weight of lm after incubation] (mg)
Incubation time (min)
Degradation of lms
in aquatic environment
Electrospun polymer lms were inserted into
four twin pouches of a nylon mesh jacket with each
– 240 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
pouch measuring 2.5 cm x 2.5 cm and with pore
diameter of 2.54 mm. The mesh jacket was tied to
holes drilled on a polyvinylchloride (PVC) core
measuring 60 cm in length and 15 cm in diameter
and dipped into a freshwater lake in Universiti
Sains Malaysia. Small holes were drilled into the
PVC core to facilitate water exchange during the
one week of study. The samples were retrieved
after one week, washed gently and allow to air-
dry before examination under Scanning Electron
Microscopy (SEM).
Scanning electron microscopy (SEM)
The morphologies of the electrospun lms
were examined by SEM (Leo Supra 50VP Field
emission, magnication of 1,000 to 50,000 times)
before and after the lms were subjected to
various degradation. The samples were mounted
on an aluminium stub and sputter-coated with
a layer of gold. Three voltages of 5 kV, 10 kV
and 15 kV were applied to obtain the images of
desired lms.
Results
Fabrication
P(3HB), P(3HB -co-25 mol % 4HB) and
P(3HB- co-75 mol % 4HB) polymers were
fabricated using electrospinning method and the
surface morphologies were observed under SEM.
Voltage of 30 kV with extrusion rate of 60 μL/
min was applied to 4 % P(3HB) and it showed
interconnected short bers that may be further
investigated to obtain brous structures (Fig. 1a).
The solution was also heated at 60 °C for 5, 10, 15
and 20 min and electrospun with the same voltage
and extrusion rate. P(3HB) polymer solution
heated for 15 min before fabrication produced
bers interconnected with bead structures
whereas the morphologies of other heating time
showed porous homogenous at structures
(Fig. 2). P(3HB) with different concentrations
were also fabricated with 15 kV and 40 μL/min.
The surface morphologies were similar but with
increasing complicacy amongst the interconnected
bers and beads as the concentration increased
from 1 % to 3 %. However, 4 % P(3HB) appeared
to be homogenous beaded structures which were
clumped together (Fig. 1b).
P(3HB- co-25 mol % 4HB) and P(3HB-co-
75 mol % 4HB) fabricated after heating at 50 °C
did not show network str ucture. Instead, P(3HB-
co-25 mol % 4HB) fabricated surface appeared to
be rough and uneven with hollow depression after
heating for 15 and 20 min (Fig. 3a & b). P(3HB-
co-75 mol % 4HB) surface showed smooth and
bead structures when heating was increased to 20
min (Fig. 3c & d). Both surface morphology of
1 % P(3HB-co-25 mol % 4HB) and 1 % P(3HB-
Fig. 1: SEM image of 4 % P(3HB) electrospun at (a)30 kV with extrusion rate 60 μL/min and (b)
15 kV with 40 μL/min of extrusion rate.
Fig. 2: SEM image of 4 % P(3HB) electrospun at 30 kV, 60 μL/min with prior heating of polymer
solution at 60 °C before fabrication for (a) 5 min; (b) 10 min; (c) 15 min; (d) 20 min.
Fig. 3: SEM image (x1000) of 1 % P(3HB-co-25 mol % 4HB) fabricated by electrospinning at 30
kV, 60 μL/min with prior heating of polymer solution at 50 °C for (a) 15 min; (b) 20 min. 1 %
P(3HB-co-75 mol % 4HB) heated at 50 °C for (c) 15 min; (d) 20 min
4
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a b
Fig. 1: SEM image of 4 % P(3HB) electrospun at (a)30 kV with extrusion rate 60 μL/min and (b) 15 kV with 40
μL/min of extrusion rate
– 241 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
Fig. 1: SEM image of 4 % P(3HB) electrospun at (a)30 kV with extrusion rate 60 μL/min and (b)
15 kV with 40 μL/min of extrusion rate.
Fig. 2: SEM image of 4 % P(3HB) electrospun at 30 kV, 60 μL/min with prior heating of polymer
solution at 60 °C before fabrication for (a) 5 min; (b) 10 min; (c) 15 min; (d) 20 min.
Fig. 3: SEM image (x1000) of 1 % P(3HB-co-25 mol % 4HB) fabricated by electrospinning at 30
kV, 60 μL/min with prior heating of polymer solution at 50 °C for (a) 15 min; (b) 20 min. 1 %
P(3HB-co-75 mol % 4HB) heated at 50 °C for (c) 15 min; (d) 20 min
4
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a b
Fig. 2: SEM image of 4 % P(3HB) electrospun at 30 kV, 60 μL/min with prior heating of polymer solution at 60
°C before fabrication for (a) 5 min; (b) 10 min; (c) 15 min; (d) 20 min
Fig. 1: SEM image of 4 % P(3HB) electrospun at (a)30 kV with extrusion rate 60 μL/min and (b)
15 kV with 40 μL/min of extrusion rate.
Fig. 2: SEM image of 4 % P(3HB) electrospun at 30 kV, 60 μL/min with prior heating of polymer
solution at 60 °C before fabrication for (a) 5 min; (b) 10 min; (c) 15 min; (d) 20 min.
Fig. 3: SEM image (x1000) of 1 % P(3HB-co-25 mol % 4HB) fabricated by electrospinning at 30
kV, 60 μL/min with prior heating of polymer solution at 50 °C for (a) 15 min; (b) 20 min. 1 %
P(3HB-co-75 mol % 4HB) heated at 50 °C for (c) 15 min; (d) 20 min
4
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6
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a b
Fig. 3: SEM image (x1000) of 1 % P(3HB-co-25 mol % 4HB) fabricated by electrospinning at 30 kV, 60 μL/min
with prior heating of polymer solution at 50 °C for (a) 15 min; (b) 20 min. 1 % P(3HB-co-75 mol % 4HB) heated
at 50 °C for (c) 15 min; (d) 20 min
co-75 mol % 4HB) were dissimilar. Surface of
P(3HB- co-75 mol % 4HB) was more rounded and
smooth compared to P(3HB-co-25 mol % 4HB)
which appeared to be rough and uneven with tiny
hollow depressions spread all over the surface
(Fig. 4).
Environmental degradation
Exposure of P(3HB), P(3HB-co-25 mol %
4HB) and P(3HB-co-75 mol % 4HB) lms
electrospun for 15 min were subjected to
environmental degradation for a period of one
week. P(3HB) with solution concentration 3 % and
4 % showed difference in morphology after a week
of immersion in the freshwater lake. Morphology
of 3 % P(3HB) changed from being intertwined
mesh bers and beads (Fig. 5a) to scattered
remains of polymer and diatoms (Fig. 5b). On the
other hand, the homogeneous hollow beads of 4 %
P(3HB) (Fig. 5c) turned into a big clump of rough
polymer with diatoms intertwined after a week
of aquatic environment exposure (Fig. 5d). The
degradation of 3 % P(3HB) was relatively better
than 4 % P(3HB). The 1 % P(3HB-co-25 mol %
– 242 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
4HB) and P(3HB-co-75 mol % 4HB) electrospun
lms also showed signs of degradation after one
week exposure in freshwater lake with P(3HB-
co-75 mol % 4HB) showing better degradation as
the lm appeared relatively clear (Fig. 6).
In vitro degradation
of electrospun P(3HB) lms
in depolymerase enzyme solution
Effects of in vitro biodegradation by PHA
depolymerase on different lm thicknesses and
concentrations of electrospun P(3HB) lms were
assessed through percentage weight loss. The 4 %
P(3HB) electrospun lms of different thickness
(determined through duration of electrospinning of
5, 10, 15 and 20 min) expressed signicant weight
loss throughout the 2 h incubation period. Films
spun for 5 and 10 min showed a gradual weight
loss as compared to electrospun lms of 15 and
20 min which exhibited a rather sharp decrease in
their weight (Fig. 7). Electrospun lms fabricated
for 15 min showed the highest degradation
Fig. 4: SEM image of P(3HB-co-4HB) fabricated by electrospinning at 30 kV and extrusion rate of
60 μL/min. (a) 1 % P(3HB-co-75 mol % 4HB) (x500); (b) 1 % P(3HB-co-25 mol % 4HB) (x500);
(c) 1 % P(3HB-co-75 mol % 4HB) (x5,000); (d) 1 % P(3HB-co-25 mol % 4HB) (x5,000).
Fig. 5: SEM image of P(3HB) subjected to environmental degradation for a period of 1 week. (a) 3 %
P(3HB) before degradation; (b) 3 % P(3HB) after degradation; (c) 4 % P(3HB) before degradation; (d) 4 %
P(3HB) after degradation.
Fig. 6: SEM image of P(3HB-co-4HB) subjected to environmental degradation for a period of 1
week. (a) P(3HB-co-25 mol % 4HB) before degradation; (b) P(3HB-co-25 mol % 4HB) after
degradation; (c) P(3HB-co-75 mol % 4HB) before degradation; (d) P(3HB-co-75 mol % 4HB) after
degradation.
40
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Fig. 4: SEM image of P(3HB-co-4HB) fabricated by electrospinning at 30 kV and extr usion rate of 60 μL/min.
(a) 1 % P(3HB-co-75 mol % 4HB) (x500); (b) 1 % P(3HB-co-25 mol % 4HB) (x500); (c) 1 % P(3HB-co-75 mol %
4HB) (x5,000); (d) 1 % P(3HB-co-25 mol % 4HB) (x5,000)
Fig. 4: SEM image of P(3HB-co-4HB) fabricated by electrospinning at 30 kV and extrusion rate of
60 μL/min. (a) 1 % P(3HB-co-75 mol % 4HB) (x500); (b) 1 % P(3HB-co-25 mol % 4HB) (x500);
(c) 1 % P(3HB-co-75 mol % 4HB) (x5,000); (d) 1 % P(3HB-co-25 mol % 4HB) (x5,000).
Fig. 5: SEM image of P(3HB) subjected to environmental degradation for a period of 1 week. (a) 3 %
P(3HB) before degradation; (b) 3 % P(3HB) after degradation; (c) 4 % P(3HB) before degradation; (d) 4 %
P(3HB) after degradation.
Fig. 6: SEM image of P(3HB-co-4HB) subjected to environmental degradation for a period of 1
week. (a) P(3HB-co-25 mol % 4HB) before degradation; (b) P(3HB-co-25 mol % 4HB) after
degradation; (c) P(3HB-co-75 mol % 4HB) before degradation; (d) P(3HB-co-75 mol % 4HB) after
degradation.
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Fig. 5: SEM image of P(3HB) subjected to environmental degradation for a period of 1 week. (a) 3 % P(3HB)
before degradation; (b) 3 % P(3HB) after degradation; (c) 4 % P(3HB) before degradation; (d) 4 % P(3HB) after
degradation
– 243 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
Fig. 4: SEM image of P(3HB-co-4HB) fabricated by electrospinning at 30 kV and extrusion rate of
60 μL/min. (a) 1 % P(3HB-co-75 mol % 4HB) (x500); (b) 1 % P(3HB-co-25 mol % 4HB) (x500);
(c) 1 % P(3HB-co-75 mol % 4HB) (x5,000); (d) 1 % P(3HB-co-25 mol % 4HB) (x5,000).
Fig. 5: SEM image of P(3HB) subjected to environmental degradation for a period of 1 week. (a) 3 %
P(3HB) before degradation; (b) 3 % P(3HB) after degradation; (c) 4 % P(3HB) before degradation; (d) 4 %
P(3HB) after degradation.
Fig. 6: SEM image of P(3HB-co-4HB) subjected to environmental degradation for a period of 1
week. (a) P(3HB-co-25 mol % 4HB) before degradation; (b) P(3HB-co-25 mol % 4HB) after
degradation; (c) P(3HB-co-75 mol % 4HB) before degradation; (d) P(3HB-co-75 mol % 4HB) after
degradation.
40
μ
m40
μ
m
40
μ
m40
μ
m
40
μ
m40
μ
m
40
μ
m40
μ
m
40
μ
m40
μ
m
40
μ
m40
μ
m
Fig. 6: SEM image of P(3HB-co-4HB) subjected to environmental degradation for a period of 1 week. (a) P(3HB-
co-25 mol % 4HB) before degradation; (b) P(3HB-co-25 mol % 4HB) after degradation; (c) P(3HB-co-75 mol %
4HB) before degradation; (d) P(3HB-co-75 mol % 4HB) after degradation
Fig. 7: Weight loss of electrospun P(3HB) films fabricated at different time durations (5, 10, 15 and
20 min) after incubation in depolymerase enzyme at 37 °C.
Fig. 8: Degradation rate of electrospun P(3HB) films fabricated at different time durations after
incubation in PHA depolymerase at 37 °C.
Fig. 9: Weight loss of P(3HB) films electrospun for 15 min using different P(3HB) concentrations
after incubation in PHA depolymerase at 37 °C.
Fig. 10: Weight loss of P(3HB-co-25 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 7: Weight loss of electrospun P(3HB) lms fabricated at different time durations (5, 10, 15 and 20 min) after
incubation in depolymerase enzyme at 37 °C
rate (Fig. 8). The degradation rate remained
almost constant as the electrospinning time was
increased to 20 min. Thus, 15 min fabrication time
was xed in subsequent experiments. P(3HB)
polymers with concentrations of 1 %, 2 %, 3 %
and 4 % which were electrospun for 15 min were
all degraded by PHA depolymerase. Electrospun
3 %, 4 % P(3HB) lms were degraded in a rather
parallel fashion showing a gradual but steady
decrease in polymer weight. The 2 % P(3HB) lm
was degraded faster compared to the 1 % P(3HB)
which exhibited slow decrease in polymer weight
(Fig. 9). There was a sharp increase in degradation
rate when electrospun P(3HB) lm concentration
shifted from 1 % (0.03 mg/min) to 2 % (0.14
mg/min). The 2 % P(3HB) lms showed the
highest degradation rate amongst the other lms
electrospun at different concentrations. After the
peak at 2 %, further increase in polymer solution
concentration resulted in a decrease of degradation
rate whereby the rate was approximately 0.1 mg/
min. Degradation rate of 3 % and 4 % P(3HB)
electrospun lms by PHA depolymerase at 37 °C
remained constant throughout the experiment.
– 244 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
In vitro degradation
of electrospun P(3HB-co-4HB) lms
in lipase enzyme solution
P(3HB- co-25 mol % 4HB) was electrospun
for 5, 10, 15 and 20 min to test the effect of layer
thickness on in vitro biodegradation using lipase
enzyme solution. The weight loss of P(3HB-co-
25 mol % 4HB) electrospun lms fabricated at
different time durations after incubation in lipase
enzyme (Fig. 10) showed that 5 min electrospun
lm started to degrade after 60 min whereas both
15 and 20 min electrospun lm started at 30 min
but with a difference in the decline pattern. The
15 min electrospun lm exhibited a big decline
in polymer weight after 60 min of incubation and
gradual decline after 90 and 120 min of incubation.
The 20 min electrospun lm was degraded after
30 min but the weight remained constant for a
period of one hour before another plunge in the
graph indicating weight loss at 120 min. The
weight decline after 30 min of incubation for the
polymer electrospun for 10 min was negligible
as the weight loss was not signicant and did not
show any further decrease in weight for another
90 min thereafter (Fig. 10). P(3HB-co-4HB) lms
electrospun for different period of time showed
Fig. 7: Weight loss of electrospun P(3HB) films fabricated at different time durations (5, 10, 15 and
20 min) after incubation in depolymerase enzyme at 37 °C.
Fig. 8: Degradation rate of electrospun P(3HB) films fabricated at different time durations after
incubation in PHA depolymerase at 37 °C.
Fig. 9: Weight loss of P(3HB) films electrospun for 15 min using different P(3HB) concentrations
after incubation in PHA depolymerase at 37 °C.
Fig. 10: Weight loss of P(3HB-co-25 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 8: Degradation rate of electrospun P(3HB) lms fabricated at different time durations after incubation in
PHA depolymerase at 37 °C
Fig. 7: Weight loss of electrospun P(3HB) films fabricated at different time durations (5, 10, 15 and
20 min) after incubation in depolymerase enzyme at 37 °C.
Fig. 8: Degradation rate of electrospun P(3HB) films fabricated at different time durations after
incubation in PHA depolymerase at 37 °C.
Fig. 9: Weight loss of P(3HB) films electrospun for 15 min using different P(3HB) concentrations
after incubation in PHA depolymerase at 37 °C.
Fig. 10: Weight loss of P(3HB-co-25 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 9: Weight loss of P(3HB) lms electrospun for 15 min using different P(3HB) concentrations after incubation
in PHA depolymerase at 37 °C
– 245 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
similar trend as P(3HB). Films electrospun for 15
min showed the highest value of degradation rate
(0.013 mg/min) and it dropped to 0.007 mg/min
for lms fabricated for 20 min (Fig. 11).
The semitransparent P(3HB-co-75 mol %
4HB) fabricated at different time durations
showed weight loss after incubation in lipase
enzyme at 37 °C (Fig. 12). Graphs of electrospun
lms fabricated at 5, 10 and 20 min showed a
rather similar pattern in polymer weight decline
throughout the 2 h of incubation. Similarly, the 15
min electrospun lms also exhibited a decrease
in its polymer weight but unlike the other lms,
this lm showed a sharp decline after 30 min
of incubation and polymer weight continued to
decrease drastically until it reached its lowest
point at 120 min. The degradation rates of P(3HB-
co-75 mol % 4HB) at 5, 10, 15 and 20 min (Fig. 13)
were similar to the degradation rate of P(3HB-co-
25 mol % 4HB) (Fig. 11) with 15 min electrospun
lm exhibiting the highest degradation rate at
0.035 mg/min followed by a decrease at 20 min
electrospun lms (0.025 mg/min).
Discussion
Fabrication
Electrospun bers have high surface area-
to-volume ratio, porous and enhanced specic
Fig. 7: Weight loss of electrospun P(3HB) films fabricated at different time durations (5, 10, 15 and
20 min) after incubation in depolymerase enzyme at 37 °C.
Fig. 8: Degradation rate of electrospun P(3HB) films fabricated at different time durations after
incubation in PHA depolymerase at 37 °C.
Fig. 9: Weight loss of P(3HB) films electrospun for 15 min using different P(3HB) concentrations
after incubation in PHA depolymerase at 37 °C.
Fig. 10: Weight loss of P(3HB-co-25 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 10: Weight loss of P(3HB-co-25 mol % 4HB) electrospun lms fabricated at different time durations after
incubation in lipase en zyme at 37 °C
Fig. 11: Degradation rate of P(3HB-co-25 mol % 4HB) electrospun film fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 12: Weight loss of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C
Fig. 13: Degradation rate of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different
time durations after incubation in lipase enzyme at 37 °C.
Fig. 14: SEM image of P(3HB-co-25 mol % 4HB) subjected to degradation by lipase enzyme
solution. (a) and (c): 1 % P(3HB-co-25 mol % 4HB) before degradation. (b) and (d): 1 % P(3HB-
co-25 mol % 4HB) after degradation.
40
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m40
μ
m
4
μ
m2
μ
m
Fig. 11: Degradation rate of P(3HB-co-25 mol % 4HB) electrospun lm fabricated at different time durations after
incubation in lipase en zyme at 37 °C
– 246 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
mechanical performance for various applications
such as ltration media, tissue engineering,
electronic applications, nanosensors and many
more (Cheng et al., 2008). Viscosity of polymer
solution is one of the key parameter that affects
ber morphology. Solution viscosity also
inuences ber diameter, initiating droplet shape
and the jet stream. Viscous polymer solution is
important in the ber forming process because
the initial jet does not break up in droplets but
instead the solvent (chloroform) will evaporate
as the jet proceeds to the target leaving behind
the polymer ber. Uniform bers will form as
the bead shape changes from spherical to thread-
like with the increase in viscosity. The presence
of repulsive Coulomb interactions in uid jet
charged elements could lead to development of
beaded structure along with the bers (Agarwal
et al., 2013). Polymer solution of 4 % P(3HB) was
heated up for 5, 10, 15 and 20 min respectively
before fabrication to increase polymer viscosity
and homogeneous mixing which could promote
ber formation (Fig. 2). The increase in polymer
concentration within the optimal concentration
range could promote formation of ber with larger
diameter (Wang et al., 2013). The 4 % P(3HB)
heated up to 15 min at 60 °C before fabrication
produced more brous network but still the
Fig. 11: Degradation rate of P(3HB-co-25 mol % 4HB) electrospun film fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 12: Weight loss of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C
Fig. 13: Degradation rate of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different
time durations after incubation in lipase enzyme at 37 °C.
Fig. 14: SEM image of P(3HB-co-25 mol % 4HB) subjected to degradation by lipase enzyme
solution. (a) and (c): 1 % P(3HB-co-25 mol % 4HB) before degradation. (b) and (d): 1 % P(3HB-
co-25 mol % 4HB) after degradation.
40
μ
m40
μ
m
4
μ
m2
μ
m
Fig. 12: Weight loss of P(3HB-co-75 mol % 4HB) elect rospun lms fabricated at different time durations after
incubation in lipase en zyme at 37 °C
Fig. 11: Degradation rate of P(3HB-co-25 mol % 4HB) electrospun film fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 12: Weight loss of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C
Fig. 13: Degradation rate of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different
time durations after incubation in lipase enzyme at 37 °C.
Fig. 14: SEM image of P(3HB-co-25 mol % 4HB) subjected to degradation by lipase enzyme
solution. (a) and (c): 1 % P(3HB-co-25 mol % 4HB) before degradation. (b) and (d): 1 % P(3HB-
co-25 mol % 4HB) after degradation.
40
μ
m40
μ
m
4
μ
m2
μ
m
Fig. 13: Degradation rate of P(3HB-co-75 mol % 4HB) electrospun lms fabr icated at different time durations
after incubation in lipase enzyme at 37 °C
– 247 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
morphology of ‘bead on a string’ was inevitable.
It was found that electrospinning from solutions
heated for more than 15 min only produced porous
interconnected mesh as homogeneous ber
formation was prohibited by high viscosity. The
highly viscous polymer solution proved difcult
to force through the syringe and thus making the
extrusion rate from the tip unstable and render
formation of bers difcult (Fig. 2d). P(3HB-co-
25 mol % 4HB) and P(3HB-co-75 mol % 4HB)
electrospun lms did not produce brous network
although the polymer solutions were heated for 15
and 20 min before fabrication. This may be due to
low viscosity of P(3HB-co-4HB) that prohibited
ber formation. Beaded structures were more
prone to form in polymer solutions fabricated at
low viscosity because droplets will be formed at
the nozzle of the syringe (Fig. 3).
Increase in polymer concentration has two
major effects on the electrospinning process
which are an increase in the evaporation rate
and ber pulling force. According to Deitzel et
al. (2001), ber diameter increases as the solution
concentration increases. Polymer concentration is
also correlated with the viscosity factor. Research
has proven that at higher concentrations of
polymer solution, bers are prone to form. This
is due to the entanglement of viscous polymer
molecule that prevents the breakup of jet directed
towards the collector (Cheng, 2008). However
in this study, 3 % P(3HB) solution showed more
signs of possible ber formation compared to 4 %
P(3H B).
P(3HB) is a stiff, crystalline and brittle
polyester. Due to these limitations, incorporation
of a second monomer, 4HB into P(3HB) polymer
backbone improves its physical characteristics.
P(3HB- co-4HB) shows properties from being
crystalline to elastic and is suitable for a wide
range of applications. One of the signicant
application is to mimic the real microenvironment
of extracellular matrix (ECM) (Li et al., 2008)
by which electrospinning could obtain three-
dimensional continuous brous network
morphology. Fibrous network changed into
porous mats with well distributed beads in
P(3HB- co-75 mol % 4HB) as the ratio of 4HB
increased (Fig. 4a & b). Thus, it is suggested that
the mixing ratio of 4HB into P(3HB) polymer
backbone to be maintained in an equilibrium of
percentage of 4HB above 25 % but below 75 % to
allow formation of a continuous brous network.
Further study is needed to establish the optimal
copolymer percentage which can produce the
desired brous matrix. Matrices of P(3HB-co-
75 mol % 4HB) showed decreased porosities
accompanied with blurred brous structures and
beaded defects. Chloroform is the only organic
solvent used in this study to dissolve polymers
as it allows full extension of the polymer and
evaporates completely after ber formation
without leaving any residue on the formed bers.
Therefore, the effect of solvent in producing bers
is not applicable here.
In vitro degradation
of electrospun P(3HB) lm
in depolymerase enzyme solution
PHA depolymerases are carboxyesterases
that hydrolyse water insoluble PHA into water-
soluble oligomers and monomers sequentially.
The monomers are then metabolized into water
and carbon dioxide or methane by other enzymes
(Amara, 2008). The enzymatic degradation of
the polymers by PHA depolymerase involves
two distinct domains. The initial stage whereby
the initial adsorption of PHA depolymerase
molecules onto the surface of P(3HB) through
enzyme substrate-binding domain (C-terminal
substrate binding domain) exhibited relatively
slow rate of weight loss as shown in Figure 7.
Adsorption is then subsequently followed by
the hydrolysis of polymer backbone through the
activity of enzyme catalytic domain (N-terminal
– 248 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
catalytic domain). Second step is essential as it is
the stage whereby the degradation of polymer by
PHA depolymerase shows rapid weight loss. The
rate of enzyme excision increases and continuous
weight loss is derived from the fragmentation of
sample and diffusion of oligomers from the bulk
polymer samples (Sudesh et al., 2000; Wang et al.,
2008). Depolymerase enzyme used in this study
was extracted from Ralstonia pickettii. Previous
studies have shown that PHA depolymerase
isolated from this bacterial species attaches rst
to the amorphous region as it is easily accessible
to enzyme compared to the crystalline structure.
The degradation rate of the polymer depends on
monomer composition and degree of crystallinity
(Li et al., 2007). Films fabricated for 5, 10, 15 and
20 min showed slow degradation initially when
incubated in the buffer solution containing PHA
depolymerase but degradation became quicker as
incubation time increased. The weight of polymer
lms increased signicantly as the electrospinning
time increased. The lm electrospun for 15 min
showed the highest degradation rate (0.1 mg/min)
(Fig. 8) and this could be because the sprayed
sample achieved the highest surface area on the
limited 1 cm x 1 cm collecting area. This enabled
more depolymerase enzymes to attach efciently.
The rate of degradation increased proportionally
with the surface area as more binding domains for
attachment of enzymes are available. Enzymatic
degradation of PHA lms was performed under
incubation at 37 °C and pH 7.4. The electrospun
lms with fabrication time of 20 min did not show
signicant increase in rate of degradation (Fig. 8).
This indicated that the prolonged electrospinning
time did not signicantly increase surface area of
the lm.
After determining the optimal electrospinning
time at 15 min, the study was preceded by
investigating the effect of P(3HB) solution
concentration on the rate of polymer degradation.
There was no clear correlation between initial
weight and the degradation rate of the polymer
because all lms were signicantly degraded
with 1 % P(3HB) being the least degraded and
2 % being the most degraded as shown in Figure
13. Slow degradation was observed initially and
thereafter degradation became faster. The highest
rate of degradation showed in 2 % P(3HB) may
be due to its higher surface area which favors the
adsorption and hydrolysis process of the PHA
depolymerase. The increase in concentration
of the electrospun lms to 3 % and 4 % did not
increase the rate further as the surface of the
polymer was probably totally saturated with
enzyme. There is no extra surface area for the
attachment of the enzymes. Another explanation
would be that there is no more available enzyme
to degrade the electrospun lms. Control in this
study showed no weight loss when immersed in
phosphate buffer without enzyme thus indicating
no signicant polymer hydrolysis occurred during
incubation in buffer solution.
In vitro degradation
of electrospun P(3HB-co-4HB) lm
in lipase enzyme solution
The degradation mechanism of lipase
enzyme on P(3HB-co-4HB) copolymer was
similar to the degradation of depolymerase
enzyme on P(3HB) homopolymer in which the
amorphous region of the copolymer was favorable
for enzyme attachment and activity (Hsieh et al.,
2006; Mitomo et al., 2001). SEM studies on both
polymers showed that the porosity of polymer
increased after degradation and indicated
successful lipase degradation on polymer surface
(Fig. 10). Earlier researches have shown that the
monomer composition and degree of crystallinity
of copolymer inuences the rate of degradation by
lipase enzyme (Hsieh et al., 2006). Therefore, an
increase in the incorporation of 4HB monomers
into 3HB backbone will increase the rate of
degradation due to lower crystallinity. Lipase
– 249 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
cleave the ester bonds of aliphatic polyesters at
the monomer sequence of (4HB)-(4HB) but not
(4HB)-(3HB). In P(3HB-co-4HB), the erosion rate
by lipase was higher with higher content of 4HB
in the copolymer (Tan, 2003). Another reason that
supports the theory that the lipase enzyme may
share a similar mechanism in substrate hydrolysis
with PHA depolymerase is that lipase also have a
common amino acid sequence around the active
side, Gly-X1-S e r-X 2-Gly, which is also observed
in PHA depolymerase. The only difference is
that the X1 residue in PHA depolymerase is
substituted with histidine in lipase (Tokiwa &
Calabia, 2004). In this study, lipase from Sigma-
Aldrich, extracted from porcine pancreas was
used in the form of lyophilized powder with an
activity of ≥20,000 units/mg protein.
Comparisons between the in-vitro
degradation of P(3HB-co-25 mol % 4HB) and
P(3HB- co-75 mol % 4HB) electrospun for 5, 10,
15 and 20 min respectively was done to elucidate
optimal electrospinning time for maximum
degradation and also to investigate the effects of
4HB ratio on the degradation rate (Fig. 10 & 13).
The two polymers exhibited increase in initial
polymer weight as the electrospinning time
increased. The highest rate of degradation was
observed in 15 min electrospinning time of P(3HB-
co-25 mol % 4HB) and P(3HB-co-75 mol %
4HB). The increase of surface area in which the
collection area was 1 cm x 1 cm was proportional
to the increase of degradation rate as there
were more surface for enzyme attachment. The
increase in electrospinning time to 20 min did not
give any signicant increase in degradation rate.
This prolonged fabrication time did not exactly
increased the surface area for enzyme attachment
but only increase the polymer weight in general.
Spherulitic textures emerging from the bulk
polymer after degradation with lipase were likely
caused by the removal of amorphous component
during degradation. The amorphous region of
polymer samples is degraded preferentially (Wang
et al., 2008). However, there are some drawbacks
in this study as agitation was not introduced for
fear of mechanical erosion of P(3HB-co-4HB)
lms. Therefore, the homogeneity of enzyme
distribution was probably compromised because
the lipase solution was only resuspended using a
pipette at the start point of the experiment.
Generally, P(3HB-co-75 mol % 4HB)
degradation was three times better than P(3HB-
co-25 mol % 4HB). This indicated that the rate of
degradation due to incorporation of higher content
of 4HB decreased the crystallinity. In P(3HB-
co-75 mol % 4HB), there was higher occurrence
of (4HB)-(4HB) monomer chains in the P(3HB)
backbone. As a result, the chances of lipase
enzymes degrading the P(3HB-co-75 mol % 4HB)
will be higher compared to P(3HB-co-25 mol %
4HB). Control in this study showed no weight
loss when immersed in phosphate buffer without
enzyme addition thus indicating no signicant
hydrolysis by buffer occurred during incubation.
Environmental degradation
PHAs have very high molecular weight and
very difcult to be transported into the cells.
Therefore, microorganisms have to excrete
extracellular PHA depolymerase to degrade
the extracellular PHA. The solid PHA can then
be hydrolysed into water soluble oligomers
and monomer which will be subsequently
utilized by the PHA degrading microorganisms
(Reddy et al., 2008). According to Mukai & Doi
(1993), PHA degrading microorganisms can
be isolated from various environments such as
soil (Pseudomonas lemoignei), activated sludge
(Alcaligenes faecalis), lab settings (Pseudomonas
pickettii), sea water (Comamonas testosterone)
and lake water (Pseudomonas stutzeri). In this
study, the degradation performance of P(3HB)
and P(3HB-co-4HB) in lake environment (fresh
water) was investigated. Liquid environment was
– 250 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
chosen because the rich microbial population or
activity of microorganisms is closely connected
to the presence of water. However, non-biotic
effects such as irradiation, thermal degradation or
chemical hydrolysis also contribute to degradation
of PHAs but at a lower rate (Miiller, 2005).
Parameters tested in this study were
concentration of PHA and effect of copolymer
on degradation. The degradation effects on
electrospun lms after immersion in lake water
were evaluated through SEM studies. 3 %
and 4 % P(3HB) were electrospun for 15 min
and immersed into the freshwater lake for a
week to compare the degradation rate. SEM
investigation of electrospun lm thereafter
revealed that degradation of the electrospun lms
of 3 % and 4 % P(3HB) occurred at the surface
by enzymatic hydrolysis. The degradation of
P(3HB- co-4HB) copolymer began with surface
erosion by microorganisms, followed by gradual
deterioration to the innermost (Weng et al.,
2013). Both lms showed signs of degradation
as the lms were eroded and diatoms were
found to remain attaching on polymer (Fig. 14).
3 % P(3HB) degraded faster compared to 4 %
P(3HB) with the assumption that environmental
conditions are constant (pH, temperature, water
activity, etc.) for both lms. Therefore, the effect
of substrate concentration on P(3HB) degradation
exhibited an optimum concentration at 3 %. The
mechanism of degradation of P(3HB) is through
ester hydrolysis at the surface. Degradation rate
of higher concentration of P(3HB) at 4 % did not
improved as the depolymerase enzyme secreted
by the microbial population was probably already
saturated. SEM studies conrmed that 4 %
P(3HB) was degraded much slower compared to
3 % P(3HB).
The second parameter investigated was
the degradation of copolymer P(3HB-co-
4HB) in environmental conditions. Research
has shown that the rate of surface erosion is
highly dependent on molecular weight, polymer
composition, crystallinity and the dominant
species of bacteria existing in the environment
(Gu, 2003). An assumption was made that
extracellular PHA depolymerases and lipases
secreted by a wide range of microbial population
in the environment can degrade P(3HB-co-
4HB). So, P(3HB-co-4HB) should show better
degradation compared to P(3HB) as P(3HB)
can only be degraded by PHA depolymerase.
Furthermore, microbes prefer to degrade co-
polymers rather than homopolymer because the
Fig. 14: SEM image of P(3HB-co-25 mol % 4HB) subjected to degradation by lipase enz yme solution. (a) and (c):
1 % P(3HB-co-25 mol % 4HB) before degradation. (b) and (d): 1 % P(3HB-co-25 mol % 4HB) after degradation
Fig. 11: Degradation rate of P(3HB-co-25 mol % 4HB) electrospun film fabricated at different time
durations after incubation in lipase enzyme at 37 °C.
Fig. 12: Weight loss of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different time
durations after incubation in lipase enzyme at 37 °C
Fig. 13: Degradation rate of P(3HB-co-75 mol % 4HB) electrospun films fabricated at different
time durations after incubation in lipase enzyme at 37 °C.
Fig. 14: SEM image of P(3HB-co-25 mol % 4HB) subjected to degradation by lipase enzyme
solution. (a) and (c): 1 % P(3HB-co-25 mol % 4HB) before degradation. (b) and (d): 1 % P(3HB-
co-25 mol % 4HB) after degradation.
40
μ
m40
μ
m
4
μ
m2
μ
m
– 251 –
Lee Joyyi, Nanthi ni Sridewi… Fabrication and Degradation of Electrospun Polyhydroxyal kanoate Film
highly crystalline structure of homopolymer
makes degradation difcult. The copolymers
exhibited higher degradation rate could also be
due to the steric hindrance presents between
carbonyl oxygen atoms of 4HB monomer
(Salim et al., 2012). From the SEM study, it
was found that the P(3HB-co-75 mol % 4HB)
was rapidly degraded compared to P(3HB-
co-25 mol % 4HB). The presence of high
percentages of 4HB monomers in the P(3HB)
backbone disrupts the crystalline str uct ure of
the homopolymer and increases the amor phous
proportion of the polymer structure. A decrease
in crystallinity aids in the degradation process
as the aquatic environment enzymes secreted
by microorganisms preferentially degrade
the amorphous part of polymers rather than
the cr ystalline region thereby increasing the
degradation rate (Sridewi et al., 2006).
Conclusion
Fabrication of P(3HB) homopolymer
using electrospinning technique produced
bers when a voltage of 30 kV was applied to
polymer solution which was extruded at a rate
of 60 μL/min. Preheating of the solution for
15 min at 60 °C had an added advantage in
improving the miscibility and homogeneity
of polymer solution prior to electrospinning.
Fabricated lm of P(3HB-co-25 mol % 4HB)
and P(3HB- co-75 mol % 4HB) copolymers
showed the best degradation rate. SEM studies
on the P(3HB) and P(3HB-co- 4HB) revealed
that P(3HB) exhibited hairy appearances after
degradation by PHA depolymerase and P(3HB-
co-4HB) became more porous after degradation
by lipase enzyme. Degradation of polymer by
environmental exposure for 1 week conrmed
that microbial activity plays important role
in the degradation of P(3HB) and P(3HB-co-
4HB). SEM study also revealed the attachment
of diatoms on the lms; therefore it is possible
that these microorganisms also contribute to the
disintegration of the polymer lms.
Acknowledgement
This research was supported by the Long
Term Research Grant Scheme (LRGS) (203/
PKT/6725001). We are grateful to J. M. M. Tay for
her helpful contribution in this research. L. Joyyi
would like to thank Universiti Sains Malaysia
LRGS for the nancial support.
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