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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
Department of Biology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
Biomaterials Science and Engineering Laboratory, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
d
Division of Health Sciences and Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
e
Engineering Systems Division Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
b
c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
a r t i c l e
i n f o
Article history:
Received 27 May 2012
Received in revised form 3 June 2012
Accepted 9 July 2012
Available online 16 July 2012
Keywords:
Cupriavidus necator
Crude palm kernel oil
Poly(3-hydroxybutyrate-co-3hydroxyhexanoate)
Bioplastics
Polymer characterization
a b s t r a c t
The potential of plant oils as sole carbon sources for production of P(3HB-co-3HHx) copolymer containing
a high 3HHx monomer fraction using the recombinant Cupriavidus necator strain Re2160/pCB113 has
been investigated. Various types and concentrations of plant oils were evaluated for efcient conversion
of P(3HB-co-3HHx) copolymer. Crude palm kernel oil (CPKO) at a concentration of 2.5 g/L was found to be
most suitable for production of copolymer with a 3HHx content of approximately 70 mol%. The time prole of these cells was also examined in order to study the trend of 3HHx monomer incorporation, PHA
production and PHA synthase activity. 1H NMR and 13C NMR analyses conrmed the presence of
P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction, in which monomers were not
randomly distributed. The results of various characterization analyses revealed that the copolymers containing a high 3HHx monomer fraction demonstrated soft and exible mechanical properties.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Polyhydroxyalkanoate (PHA) is typically present in cells in the
form of hydrophobic inclusion bodies, which are produced when
nutrients required for growth, such as nitrogen, magnesium, sulfur
or phosphorus, are limited. Some bacteria are capable of accumu Corresponding author. Tel.: +60 4 6534367.
E-mail address: ksudesh@usm.my (K. Sudesh).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.07.015
general, most PHA that has been extensively studied has been classied into three main types, depending on the number of carbon atoms
in the monomer units. PHA containing monomers consisting of 35
carbon atoms are called short chain length PHA (scl-PHA); polymer
with monomers consisting of 614 carbon atoms are mediumchain-length PHA (mcl-PHA); and copolymer containing combinations of scl- and mcl-PHA monomers are referred to as mixed chain
length PHA (Madison and Huisman, 1999). There are some physical
differences among scl- and mcl-PHA. The mcl-PHA polymers are typically sticky, elastic, and amorphous materials, while scl-PHA are
highly crystalline thermoplastic materials (Sudesh et al., 2000). Also,
for scl-PHA, the monomer units can be oxidized at positions other
than the third carbon, while for mcl-PHA, monomer units, with few
exceptions, are typically oxidized at the third position.
P(3HB) is the most common type of PHA found in nature, and it
is more crystalline than PHA copolymers, with applications normally limited to the production of thermoplastic (Sudesh et al.,
2000). P(3HB-co-3HHx) is a type of copolymer which can be
produced by some wild-type strains of bacteria, for example, Aeromonas caviae. PHA copolymers containing 3HB with a small
amounts of other monomer units are more exible than P(3HB)
homopolymer. Unlike P(3HB), P(3HB-co-3HHx) is a exible material and it shows a high degree of elongation to break (Doi et al.,
1995). This copolymer is suitable to be used as a lm due to its
exibility. In general, researchers have shown that the different
physical and mechanical properties of the polymer (from hard
crystalline polymer to elastomeric rubber) depend on the types
and quantities of incorporated monomeric units (Doi et al., 1995).
Plant oils have been shown to be better carbon sources for
growth and PHA accumulation than sugars for select bacteria,
including C. necator (Budde et al., 2011; Kahar et al., 2004). Plant
oils contain a higher carbon content per weight than sugars, suggesting that the theoretical yield of PHA from plant oils could be
at least twofold higher than that from sugars (Akiyama et al.,
2003).
Palm oil is a promising carbon source for microbial PHA production, and an important natural resource and commodity for Southeast Asian countries, such as Malaysia. Over the past several
decades, the oil palm industry in Malaysia has grown rapidly and
spurred economic growth. Malaysia has become one of the leading
producers and exporters of palm oil in the world today, exporting a
total of 16.7 million tonnes of palm oil into the international market in 2010. The 2010 Malaysian export of all palm oil products
including palm oil, palm kernel oil, palm kernel cake, oleochemicals, biodiesel and nished products has reached 23.1 million tonnes (MPOB, 2010). Therefore, the commercialization of PHA
production in Malaysia using palm oil as the sole carbon sources
is very promising.
C. necator (also known as Ralstonia eutropha) is a Gram-negative
betaproteobacterium that is a model PHA-producing organism
capable of accumulating PHA at high levels, exceeding 80% of dried
cell mass. However, it can produce only P(3HB) homopolymer from
simple carbon sources such as fructose. Rhodococcus aetherivorans
is a non-spore forming, Gram-positive aerobic actinomycete that
can produce PHA copolymer using sugar as the sole carbon source
(Hori et al., 2009), but it has been shown to accumulate low levels
of intracellular PHA (<2 wt.%). R. aetherivorans I24 was rst isolated
from hydrocarbon-contaminated soil for the synthesis of indinavir
sulfate, CRIXIVAN, which is a protease inhibitor used in the treatment of AIDS (Buckland et al., 1999). Budde et al. had successfully
engineered a recombinant strain of C. necator, harboring a polyhydroxyalkanoate synthase gene from R. aetherivorans strain I24
(phaC2Ra), that demonstrated remarkable enhancement in P(3HBco-3HHx) productivity (Budde et al., 2011).
In the present study, PHA containing as high as 70 mol% 3HHx
monomer content was accumulated from this recombinant
321
322
323
3HHx
CPKO
JO
CPO
PO
SBO
CRO
CO
4.96b 0.29
4.14a 0.09
4.21a 0.30
4.24a 0.34
4.56ab 0.11
4.29a 0.14
4.23a 0.19
77c 3
62ab 3
69abc 6
61a 4
65ab 1
63ab 2
70bc 3
3.8c 0.2
2.5a 0.2
2.9ab 0.0
2.6ab 0.4
3.0ab 0.1
2.6ab 0.2
3.3bc 0.4
44
59
59
57
55
54
38
56c
41a
41a
43ab
45ab
46b
62d
CPKO, crude palm kernel oil; JO, jatropha oil; CPO, crude palm oil; PO, palm olein; SBO, soybean oil; CRO, corn oil; CO, coconut oil; 3HB, 3-hydroxybutyrate monomer and
3HHx, 3-hydroxyhexanoate monomer.
Data shown are the means of triplicate tests. Mean data accompanied by different alphabet letters are signicantly different (Tukeys HSD test, p < 0.05).
*
Cells were cultivated in 50 mL MM supplemented with 5 g/L of respective carbon sources for 48 h at 30 C, 200 rpm in a 250 mL ask.
**
Cell dry weight after freeze-drying.
2012). These ideal fatty acids for growth of C. necator were found
to be abundant in CPKO. Furthermore, linolenic acid was found
only in trace amounts.
Meanwhile, 2.5 g/L of coconut oil showed the highest 3HHx
monomer fraction of the copolymer, 70 mol%. The 3HHx monomer
fraction of P(3HB-co-3HHx) decreased to 56 mol% when 5.0 g/L of
coconut oil was added into the medium but increased to 62 mol%
as the oil concentration increased to 20.0 g/L. The cell dry weight
exhibited no signicant changes as the concentration of coconut
oil increased from 2.5 to 20.0 g/L. The PHA content of the cells increased from 48 to 79 wt.% as the concentration of the supplied
carbon source increased, similar to results seen in CPKO cultures.
Generally, both CPKO and coconut oil support the synthesis of
PHA with similar 3HHx compositions, approximately 70 mol%, at
the same concentration of carbon source, 2.5 g/L. However, the
results showed that CPKO, when fed to the initial culture in
the 512.5 g/L range, could support better bacterial growth when
compared to coconut oil, as well as high PHA production (Table
2). C. necator strain Re2160/pCB113 showed high P(3HB-co3HHx) copolymer productivity concomitant with better overall
growth when CPKO was used as the sole carbon source. Also, palm
oil is more readily available in Malaysia as compared to the availability of coconut oil making it more attractive as a carbon source
for PHA production. The advantage of Malaysia as the leading producer and exporter of palm oil in the world today allows resources
to be allocated for PHA production from such oils, as well as commercialization and further investigations on polymer production.
Generally, CPKO as the sole carbon source for PHA synthesis is feasible, sustainable, and yields more encouraging results compared
to other oils. Therefore, CPKO was selected for further experiments.
3.3. Time prole for the production of P(3HB-co-3HHx) by C. necator
Re2160/pCB113
After selecting a suitable carbon source and concentration for
the production of P(3HB-co-3HHx), a time prole of copolymer biosynthesis using supplementation of 2.5 g/L CPKO was carried out,
in order to study the trend of 3HHx incorporation and overall
PHA production over time. For this, cells were harvested at intervals of every 12 h. As shown in Fig. 1, the PHA content of the cells
did not exhibit signicant differences throughout the cultivation.
These data show that this recombinant strain has favorable PHAaccumulating ability, as it demonstrated high PHA content at the
early stationary growth phase. There were few signicant changes
in cell dry weight after the rst 24 h until the end of the cultivation
period (72 h). The 3HHx monomer fraction of the copolymer was
the highest at 12 h (70 mol%) and decreased by 24 h and then
324
Table 2
Biosynthesis of P(3HB-co-3HHx) containing high 3HHx monomer fraction using CPKO or coconut oil as the sole carbon source.
Different concentrations of
oil* (g/L)
CPKO
Cell dry weight
(g/L)
PHA content
(wt.%)
Total PHA
(g/L)
Monomer
composition
(mol.%)
3HB
3HHx
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
2.77a 0.27
5.08b 0.32
6.73b 0.59
6.27b 0.55
5.41b 0.70
2.96a 0.10
2.97a 0.82
2.78a 0.29
45a 1
74b 4
83cd 3
82bcd 2
81bcd 5
88d 1
79bc 2
87cd 2
1.3a 0.1
3.7bc 0.2
4.4bc 1.9
5.1c 0.3
4.3bc 0.9
2.6ab 0.1
2.4ab 0.6
2.4ab 0.2
32
44
45
43
45
43
42
44
68b
56a
55a
57a
55a
57a
58a
56a
CO
**
PHA content
(wt.%)
Total PHA
(g/L)
2.61a 0.24
2.71a 0.15
3.48a 0.21
3.44a 0.77
3.59a 1.19
4.49a 0.54
3.93a 0.40
3.95a 0.10
48ab 3
63abc 7
63bc 3
61abc 6
59a 20
74cd 6
75cd 7
79d 2
1.3a 0.2
2.3a 0.2
2.2a 0.2
2.1a 0.7
2.3a 1.6
3.3a 0.3
3.4a 0.0
3.3a 0.3
Monomer
composition
(mol.%)
3HB
3HHx
30
44
44
41
40
38
39
38
70c
56a
56a
59ab
60ab
62b
61ab
62b
CPKO, crude palm kernel oil; CO, coconut oil; 3HB, 3-hydroxybutyrate monomer and 3HHx, 3-hydroxyhexanoate monomer.
Data shown are the means of triplicate tests. Mean data accompanied by different alphabet letters are signicantly different (Tukeys HSD test, p < 0.05).
*
Cells were cultivated in 50 mL MM supplemented with different concentrations of carbon sources for 48 h at 30 C, 200 rpm in a 250 mL ask.
**
Cell dry weight after freeze-drying.
325
326
Table 3
Gel permeation chromatography (GPC) analysis and thermal properties of P(3HB-co-3HHx) copolymer containing high 3HHx monomer fraction.
Sample
P(3HB)
P(3HB-co-32%
P(3HB-co-43%
P(3HB-co-56%
P(3HB-co-60%
P(3HB-co-70%
3HHx)
3HHx)
3HHx)
3HHx)
3HHx)
Mw (105 Da)
Mn (105 Da)
Mw/Mn
Tg* (C)
Tm* (C)
DHm** (Jg1)
Td* (C)
References
9.0014.00
3.47c 0.18
1.17a 0.06
1.20a 0.10
2.11b 0.13
2.27b 0.28
1.667.37
2.24 0.20
0.72 0.05
0.82 0.06
1.26 0.03
1.37 0.16
1.72.9
1.55ab 0.06
1.63bc 0.03
1.45a 0.01
1.75c 0.07
1.66bc 0.03
4
1
4
6
11
12
180
88
86
N.D
N.D
N.D
6080
N.D
N.D
N.D
N.D
N.D
287
278
285
274
278
278
(Doi, 1990)
This study
This study
This study
This study
This study
Mw, weight-average molecular weight; Mn, number-average molecular weight; Mw/Mn, polydispersity index; Tg, glass transition temperature; Tm, melting temperature; Td,
decomposition temperature; DHm, enthalpy of fusion and N.D, not detected.
Data shown are the means of triplicate tests. Mean data accompanied by different alphabet letters are signicantly different (Tukeys HSD test, p < 0.05).
*
Measured by differential scanning calorimetry (DSC).
**
Measured by thermogravimetric (TGA) analysis. Temperature was measured at 5% weight loss.
Table 4
Mechanical properties of P(3HB-co-3HHx) containing high 3HHx monomer fraction as measured by tensile tester.
Sample
P(3HB)
P(3HB-co-32%
P(3HB-co-43%
P(3HB-co-56%
P(3HB-co-60%
P(3HB-co-70%
LDPE
3HHx)
3HHx)
3HHx)
3HHx)
3HHx)
References
43
8d 1
5c 1
1b 1
<1b 1
<1a 1
15.278.6
3.5
101c 6
75b 9
12a 2
3a 1
<1a 0
50100
5
856b 21
481a 47
368a 1
424a 23
1075b 158
700
(Doi, 1990)
This study
This study
This study
This study
This study
(Doi, 1990)
327
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