Bioresource Technology 102 (2011) 6625–6628

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Bioresource Technology
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Short Communication

Characterisation of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate)

(PHB-co-PHV) produced by Halomonas campisalis (MCM B-1027),
its biodegradability and potential application
Snehal O. Kulkarni a, Pradnya P. Kanekar a,⇑, Jyoti P. Jog b, Prashant A. Patil b, Smita S. Nilegaonkar a,
Seema S. Sarnaik a, Pranav R. Kshirsagar a
a
Microbial Sciences Division, MACS-Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India
b
National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India

a r t i c l e i n f o a b s t r a c t

Article history: Characterisation of polyhydroxyalkanoate (PHA) film produced by haloalkalitolerant Halomonas

Received 29 December 2010 campisalis (MCM B-1027) in 14 L SS fermenter revealed it to have composition of monomer units, HB:HV
Received in revised form 16 March 2011 as 96:4 as analysed by 1H NMR indicating the PHA as a co-polymer of PHB-co-PHV, molecular weight by
Accepted 17 March 2011
gel permeation chromatography as 2.08  106, melting temperature 166.51 °C, tensile strength 18.8 MPa;
Available online 22 March 2011
two relaxations namely beta transition corresponding to the glass rubber transition and alpha transition
corresponding to crystalline relaxation by Dynamic Mechanical Thermal analysis and only one relaxation
Keywords:
corresponding to MWS interfacial polarisation with activation energy of 129 kJ/mol by broadband dielec-
Halomonas
Biodegradable polymer
tric spectroscopy. Optical microscopic studies showed typical Maltese-cross pattern of spherulites. The
Copolymer PHB-co-PHV PHA film was found to be biodegradable by standard ASTM method as well as by soil burial method.
Dynamic mechanical analysis The leak proof polymer bags prepared from the film could be used as a packaging material.
Dielectric relaxation spectroscopy Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction 2. Methods

Polyhydroxyalkanoates (PHAs) are the polyesters of various
hydroxyalkanoates which are synthesized by some microorgan-
isms as an energy reserve material. The structure and properties
of PHA films formed by either conventional solvent casting tech-
nique or melt press technique have been investigated extensively
with respect to their crystallisation behaviour (Don et al., 2006),
mechanical properties like tensile strength (Miao et al., 2008), dy-
namic mechanical analysis (Pederson et al., 2006) dielectric relax-
ation spectroscopic studies (Sics et al., 2001) etc. Degradation
studies of the PHA film formed have been investigated by some
researchers in various natural environments such as soil (Mergaert
et al., 1993) and in compost (Mergaert et al., 1994). The character-
istic properties of any polymer determine its processibility and
application. Kulkarni et al. (2010) have reported flask culture pro-
duction of PHA by Halomonas campisalis MCM B-1027. The present
paper describes characterisation of the polymer produced by the
same organism and its potential application.
2.1. Bacterial strain and production of PHA
H. campisalis MCM B-1027 (Kulkarni et al., 2010) was employed
for the production of PHA in 14 L stainless steel (SS) fermenter
(Biochem Bioengineering Pvt., Ltd., Pune) with a working volume
of 10 L. The production medium (pH 9.5) described earlier
(Kulkarni et al., 2010) was modified for sodium chloride content
as 5 g/L instead of 45 g/L and sterilised in situ for 30 min. Maltose
and calcium chloride solutions were autoclaved separately and
aseptically transferred into the fermenter. The seed inoculum of
H. campisalis prepared by growing the culture in 400 ml nutrient
broth of pH 9.0 (Kulkarni et al., 2010) having 109 cells/ml was asep-
tically transferred at 4% (v/v) to the fermenter. The fermentation
conditions were aeration 0.5 vvm initially till 12 h followed by
1.0 vvm till 24 h, agitation speed 100 rpm and temperature as
30 °C. The cells were stained with 1% (w/v) Nile Blue Sulphate
and examined under phase contrast microscope equipped with
UV (Nikon, Japan) for detection of PHA granules accumulated with-
in cells (Ostle and Holt, 1982).

2.2. Extraction of PHA

⇑ Corresponding author. Tel.: +91 020 25674995/53680; fax: +91 020 25651542.
E-mail addresses: kanekarpp@gmail.com, kanekarp@rediffmail.com (P.P. Kane- After 24 h of incubation, the cell mass was harvested by using
kar). continuous centrifuge (Pennwalt, Mumbai, India), dried using

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.03.054
6626 S.O. Kulkarni et al. / Bioresource Technology 102 (2011) 6625–6628
lyophilizer and the weight of dry cell mass was noted (Kulkarni
et al., 2010). PHA was extracted in chloroform from dried cell mass
using rotary film evaporator under vacuum for 3 h. After extrac-
tion, the PHA obtained was purified by methanol precipitation
(Kulkarni et al., 2010). The white precipitate of PHA was used to
cast the films by conventional solvent cast technique for further
studies.
2.3. Characterisation of PHA film
The PHA film obtained was characterised for (i) composition of
monomer units by 1H NMR analysis (Bloembergen and Holden,
1986); (ii) molecular weight by gel permeation chromatography
and melting temperature by differential scanning calorimetric
analysis (Kulkarni et al., 2010); (iii) wide angle X-ray diffraction
using Pan Analytical’s Powder XRD Xpert – 1712 with Cu Ka radi-
ation (k = 1.54 Å) (Yamanea et al., 2001); (iv) spherulitic morphol-
ogy using polarised light optical microscope (Leica Laborlux 12
pols Germany) (Don et al., 2006); (v) microtensile testing using
Linkam TST 350 stage microtensile testing machine (Misra
Fig. 1. 1H NMR spectra of PHA. (A) Standard copolymer PHB-co-PHV 9:1. (B) PHA film produced by H. campisalis showing the presence of monomer units HB and HV.
et al., 2006); (vi) Dynamic Mechanical Thermal analysis (DMTA)
using a Dynamic Mechanical Thermal analyzer (Rheometric Scien-
tific Inc., model IIIE) in linear viscoelastic region at a frequency of
10 rad/s and temperature ranging from 100 to 130 °C with
0.02% strain; (vii) dielectric spectroscopic measurement using
an active BDS cell and alpha – a high performance frequency ana-
lyzer from Novocontrol, Germany for modulus00 and conductivity’
at temperatures ranging from 30 to 130 °C and over a frequency
range of 106 to 102 Hz.
2.4. Biodegradability testing of PHA film
Biodegradability testing was performed by (i) modified stan-
dard ASTM method (D5209-91) by growing five soil isolates viz.
Bacillus subtilis (MCM B-310), Pseudomonas putida (MCM B-408),
Micrococcus lylae (MCM B-362), Arthrobacter atrocyaneus (MCM
B-425), H. campisalis (MCM B-1027) and consortium of all above
cultures in the Davis Mingioli’s minimal medium (DM) containing
0.1% (w/v) PHA film as a sole source of carbon in sealed bottles and
testing utilisation of PHA film with simultaneous evolution of car-
 S.O. Kulkarni et al. / Bioresource Technology 102 (2011) 6625–6628 6627

bon dioxide. CO2 was estimated using gas chromatograph (Chemi-
to HT 8610, Mumbai, India) equipped with thermal conductivity
detector (TCD), Porapak Q column and hydrogen gas at the rate
of 29 ml/min as a carrier and at temperatures of oven, injector
and detector as 35, 42 and 54 °C, respectively. (ii) Soil burial meth-
od: 20 lm thick PHA films (9  6 cm) were buried in the garden
soil having moisture content 15%, 20%, 25% and 30%, placed in
the plastic container and incubated at ambient temperature
(28 ± 2 °C). The biodegradation was monitored after every 7 days
interval till 2 months by measuring loss in weight of PHA films.
2.5. Potential application as packaging material
PHA films having 30 lm thickness were used for preparation of
polymer bags having dimensions (5  7.5 cm) and weight about
500 mg. The bags prepared were filled with grocery items having
weight ranging from 15 to 35 g. The bags were sealed and leakage
of the bags was checked manually.
3. Results and discussion

3.1. Production of PHA

0.08 From 10 L fermented broth, 12.14 g dry cell mass was obtained
0 which yielded 4.47 g PHA i.e. 36.82% on dry cell weight basis.
70 C
0
80 C
0
0.06 90 C 3.2. Characterisation of PHA film
0
100 C
0
110 C
(i) 1H NMR analysis revealed that PHA produced by H. campisalis
Modulus"

0
120 C
0.04 0 was a copolymer of PHB and PHV (Fig. 1B) with the molar percent-
130 C
age of HB unit as 96 and HV unit as 4 when compared to 1H NMR of
standard PHB-co-PHV 9:1 (Fig. 1A). (ii) Gel permeation chromatog-
0.02 raphy analysis showed that polydispersity index (D) was 1.25,
number average molecular weight (Mn) 1.66  106 and weight
average molecular weight (Mw) 2.08  106 which are comparable
0.00 with that of standard PHB-co-PHV 9:1 i.e. Mw 6.55  105., Mn
10
-2 -1
10
0
10 10
1
10
2
10
3
10
4 3.55  105 and polydispersity index (D) 1.84. Sudesh et al. (2000)
Frequency (Hz) reported that Mw of the PHB produced by native bacteria is usually
in the range of 5–10  105. Thus our finding matches with the re-
Fig. 2. Modulus00 as a function of frequency at different temperature for PHA film. port of Sudesh et al. (2000). The melting temperature of the PHA

Fig. 3. Biodegradability testing of PHA film. (A) Carbon dioxide evolution by different test microorganisms during biodegradation of PHA by modified ASTM method. (B)
Comparative data of effect of moisture content of soil on degradation of PHA film.
6628 S.O. Kulkarni et al. / Bioresource Technology 102 (2011) 6625–6628
film formed by H. campisalis was 166.51 °C which was lower than
standard PHB (172 °C), whereas the melting temperature of the
standard PHB-co-PHV 9:1 was much lower (143 °C). Yoshie et al.
(2004) have noted that melting temperature of PHB-co-PHV
decreased with increasing HV content. (iii) Wide angle X-ray dif-
fraction describes the crystalline structure of PHB-co-PHV co-poly-
mer produced by H. campisalis. According to Yamanea et al. (2001)
PHB is crystallised in orthorhombic unit cell with lattice parameter
a = 0.576 nm, b = 1.320 nm, c = 0.596 nm. PHB-co-PHV up to
40 mol% HV crystallised in PHB type lattice (Yoshie et al., 2001).
PHB shows characteristic reflection at 2h = 13, 16, 19.7, 25.6 and
27. Similar diffraction peaks are also observed for PHB-co-PHV
copolymer produced by H. campisalis. (iv) Mechanical testing: ten-
sile properties of the PHA films exhibited tensile strength of
18.8 MPa, elongation to break of 2.34% and tensile modulus of
1.2 GPa. These values are in good agreement with those reported
by Misra et al. (2006). (v) Optical microscopic analysis: PHA film
exhibited banded spherulites similar to that observed in PHB. A
typical Maltese-cross pattern of PHA spherulites was observed.
Similar observation was noted by Don et al. (2006). (vi) Dynamic
Mechanical Thermal analysis (DMTA): PHB-co-PHV shows crystal-
line relaxation (ac) and second relaxation i.e. b relaxation is attrib-
uted to the glass to rubber transition in the polymer. PHA produced
by H. campisalis shows alpha crystalline relaxation at 100 °C and
glass transition temperature at around 7.97 °C. Scandola et al.
(1988) reported the glass transition of PHB in the range of
0–20 °C which is comparable to our results.
(vii) Dielectric spectroscopy: the permittivity of PHA film is 4.7
at 100 Hz and at frequency 0.01 Hz which shows dielectric constant
of 8.1. The increase in dielectric constant at low frequency is gener-
ally ascribed to the conductive/Maxwell Wagner Sillar (MWS)
relaxation. Fig. 2 shows the frequency dependence of modulus00 at
various temperatures. The peak appearing at characteristic fre-
quency infers the presence of relaxatory modes in the polymer film.
This relaxation observed at low frequency side is conductive relax-
ation attributed to MWS interfacial polarisation. The loss modulus
peak shifts to the higher frequency with increase in temperature.
The activation energy was calculated as 129 kJ/mol which matches
with the activation energy calculated by Chambers et al. (2001) for
MWS interfacial polarisation.
3.3. Biodegradability testing of PHA film
(i) Standard ASTM method: in the present study, all test cultures
could utilise PHA film as a sole carbon source as there was increase
in CO2 production from Day 0 till Day 42 (Fig. 3A) indicating biode-
gradable nature of the PHA film. Kim et al. (1999) reported the
modified strum test utilising single isolated strain, an activated
sludge or synthetic sludge for measuring biodegradability of PHB
and PHB blends. (ii) Soil burial method: the rate of degradation
of the PHA film was 12% and 23.6% within 8th week respectively
in soil containing 15% and 20% moisture while in the soil having
25% and 30% moisture, the PHA films were found to be degraded
into fine pieces in 6 weeks resulting into 95% loss in weight of
PHA film (Fig. 3B) indicating the importance of moisture content
of the soil. Similar observation was noted by Wang et al. (2005)
and Woolnough et al. (2010). This confirms biodegradable nature
of PHA.
3.4. Potential application as packaging material
The sealed bags and carry bags were found to be leak proof. No
mechanical damage was observed during the ambient (28 ± 2 °C)
storage of the polymer bags over a period of 6 months. Thus the
PHA obtained from H. campisalis could be used as a packaging
material.
4. Conclusions
The polymer produced by H. campisalis was found to have ten-
sile strength of 18.8 MPa, melting temperature 166.51 °C, composi-
tion of HB and HV unit as 96 and 4 mol% respectively indicating
that the polymer was a copolymer, PHB-co-PHV and molecular
weight (Mw) of 2.08  106. The copolymer is biodegradable in nat-
ure. These properties indicated possible processing and application
of the PHA as packaging material.
Acknowledgement
The research was financially supported by Department of Bio-
technology, Govt. of India.
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