International Journal of Biological Macromolecules 46 (2010) 255–260

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Polyhydroxyalkanoate (PHA) synthesis by Spirulina subsalsa

from Gujarat coast of India
Anupama Shrivastav, Sanjiv K. Mishra, Sandhya Mishra ∗
Central Salt and Marine Chemicals Research Institute, Council for Scientific and Industrial Research, G.B. Marg, Bhavnagar 364021, India

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

Article history: Cyanobacteria have many unexploited potential for natural products with a huge variability in structure
Received 24 November 2009 and biological activity. Their products are species specific and substrate + growth condition specific. Under
Received in revised form stress conditions they are reported to produce biopolymers like EPS and PHA, which can be produced
31 December 2009
extracellularly and intracellularly, respectively. Polyhydroxyalkanoates are polymers of biological origin,
Accepted 4 January 2010
they are also capable of being completely broken down to water and carbon dioxide by microorganisms
Available online 11 January 2010
found in a wide range of environments, such as soil, water, and sewage.
We have studied marine cyanobacteria Spirulina subsalsa from Veraval coast, Gujarat, India, producing
Keywords:
Biodegradable
PHA under increased sodium chloride (NaCl) concentration (5% enhancement to the ASNIII medium), The
Cyanobacteria biopolymer was chemically characterized through FTIR, NMR, TGA, and DSC. The present study shows
PHA increased PHA accumulation in S. subsalsa by twofold increased NaCl concentration in the growth media.
Spirulina subsalsa
Sodium chloride © 2010 Elsevier B.V. All rights reserved.

1. Introduction PHB by oxygenic photosynthesis [9], and are of particular inter-

Polymers originating from living organisms are termed biopoly-
mers, which is different from manmade synthetic polymers such
as PVC and polypropylene. Biopolymers e.g. PHA can be pro-
duced from natural substrates and are 100% biodegradable. PHA
is polyester like, synthesized and stored intracellularly by bacteria
as energy and carbon storage materials having structural proper-
ties similar to polypropylene [1]. Many microorganisms synthesize
and accumulate PHA in the form of water insoluble granules [2,3].
Since, PHAs are of biological origin, they are also capable of being
completely broken down to water and carbon dioxide by microor-
ganisms found in a wide range of environments, such as soil, water,
and sewage [4–7]. PHAs have immense applications in packaging
films, disposable items, bone replacements, blood vessel replace-
ments and scaffold material in tissue, engineering of heart valves,
etc.
More than 300 different microorganisms are known to syn-
thesize and accumulate PHA intracellularly [8]. Cyanobacteria
however, are indigenously the sole prokaryotes that accumulate
∗ Corresponding author at: Marine Biotechnology and Ecology Discipline, Cen-
tral Salt & Marine Chemicals Research Institute, Council for Scientific and Industrial
Research, G.B. Marg, Bhavnagar 364021, India. Tel.: +91 278 2561354;
fax: +91 278 2567562.
E-mail addresses: smishra@csmcri.org, smishracsmcri@rediffmail.com
(S. Mishra).
est because of their minimal requirements for their growth and
biomass production [10–13]. Cyanobacteria are the oldest oxy-
genic photosynthetic prokaryotic microorganisms on the earth.
Cyanobacteria are found in almost every conceivable habitat, from
oceans to fresh water to bare rock to soil. Some cyanobacteria can
produce and accumulate PHB when grown mixotrophically with
acetate, the maximum value has been recorded for Synechocys-
tis PCC 6803 (15%, w/w dry cells) [14]. PHB production using CO2
as a carbon source by cyanobacteria is available but the contents
in general, are very low, with sole exception to Synechococcus sp.
MA19 where a higher PHB content, 27% (w/w) dry cells, has been
reported [9].
PHAs are microbial polyesters, synthesized by numerous
microorganisms having dual function as a reserve compound and
as a stress metabolite accumulating in response to stress condition.
One of the major drawbacks of employing PHA in a wide range of
applications is its high production cost. Consequently, much effort
has been devoted to reduce the production cost of PHA by improv-
ing bacterial strain, efficient fermentation and recovery processes
[5]. The stress condition of salinity in which this organism grows
almost overrides the contamination problem and can reduce the
sterility requirements of a production facility and, thus, decreases
the investment cost. Moreover, the exploitation of such cyanobac-
teria can also be done for PHA production from sludge, industrial
effluent or other wastewater which contains high salt concen-
tration. The ability of cyanobacteria to thrive in salinity makes
them candidates for industrial and bioremediation applications.

0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2010.01.001
256 A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260

The objective of the present study was to investigate the effect of
increased salinity on PHA production in Spirulina subsalsa.
2. Experimental
2.1. Test organisms and experimental conditions
Culture of S. subsalsa was grown in 250 mL Erlenmeyer flasks
containing 100 mL of modified ASNIII media at 24 ◦ C under illu-
mination with cool white fluorescent light and light–dark cycle
of 14/10 h. The media constituents were in g/L: NaCl 25.0 g,
MgCl2 ·6H2 O 2.0 g, KCl 0.5 g, NaNO3 0.75 g, K2 HPO4 ·3H2 O 0.02 g,
MgSO4 ·7H2 O 3.5 g, CaCl2 ·2H2 O 0.5 g, citric acid 0.003 g, ferric
ammonium citrate 0.003 g, EDTA (disodium magnesium salt)
0.0005 g, Na2 CO3 0.02 g, 1 mL trace metal mix A5 + Co and pH 7.5
after autoclave.
The cells grown in ASNIII media for 10 days were transferred
to nitrogen free ASNIII media (media without NaNO3 ), and having
fivefold increased NaCl concentration. The NaCl concentration was
increased by adding double the amount of NaCl salt in the media,
5% NaCl concentration in ASNIII media for S. subsalsa.
mixture was decanted and the precipitated polymer was separated
by centrifugation. Then, the polymer was dissolved in chloroform.
After evaporation of the solvent, PHB was obtained as a tough,
translucent film. Quantitative determination of PHB was done by
Law and Slepecky method [15] and gravimetrically.
2.5. Fourier transform infrared spectroscopy (FTIR)
KBr pellet was prepared using PHA from Spirulina culture and
standard PHA from Sigma. A PerkinElmer spectrum GX FTIR spec-
trometer was used with spectral range, 4000–400 cm−1 to record
the IR spectra.
2.6. Nuclear magnetic resonance (NMR)
1H NMR spectra was acquired by dissolving the polymer in
deuterochloroform (CDCl3 ) at a concentration of 10 mg/mL and
analyzed on a Bruker Avance II 500 spectrometer at 22 ◦ C with
7.4 ms pulse width (30◦ pulse angle), 1 s pulse repetition, 10,330 Hz
spectral width, 65,536 data points. Tetramethylsilane was used as
an internal shift standard.

2.2. Microscopy 2.7. Differential scanning calorimetry (DSC) and

thermogravimetry analysis (TGA)
The morphology of S. subsalsa cells was studied using scan-
ning electron microscope and light microscope equipped with The thermal properties of the cyanobacterial polyesters were
oil immersion objective (100× magnification, AXIO IMAGER, Carl examined by a DSC and TGA instrument. DSC analysis of sample
Zeiss). was done using a Mettler Toledo 822c instrument with starc soft-
For scanning electron microscopy, a small amount of the cul- ware. The calorimeter had one sample cell and one reference cell.
ture was placed on sample holder and the cells were allowed to Samples (less than 10 mg) were exposed to a temperature profile
settle for about 1 h at R.T. The samples were fixed overnight at R.T. over −30 ◦ C to 200 ◦ C, at a heating rate of 10 ◦ C min−1 for first run.
with 2% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer at The sample was cooled rapidly by quenching in liquid nitrogen,
pH 7.5. The samples were then, washed with 0.1 M sodium phos- and then analyzed again during a second heating scan from −30 to
phate buffer, pH 7.5, at room temperature for 1 h. Post-fixation was 450 ◦ C at a heating rate of 2 ◦ C min−1 .
carried out in 2% (w/v) osmium tetroxide in the same buffer and Thermal stability of the PHA was investigated by thermogravi-
washed once with 0.1 M sodium phosphate buffer for 20 min. Then, metric analysis (TGA) using a Mettler Toledo TGA/SDTA851c with
the water was removed by water–ethanol series—25% ethanol – starc software instrument under nitrogen atmosphere. The temper-
15 min, 50% ethanol – 15 min, 75% ethanol – 15 min, 90% ethanol ature range was from 30 to 500 ◦ C with a heating rate of 10 ◦ C min.
– 15 min, absolute alcohol – 15 min. The specimens were rinsed
in buffer and coated with gold in a sputtercoater (Polaron SC7620)
3. Results and discussion
prior to microscopy. The material was examined in a scanning elec-
tron microscope (SEM) LEO 1430 VP at an accelerating voltage of
3.1. Morphological and growth characteristics
15 kV.
PHA accumulation in S. subsalsa cells was observed by staining
The morphology of the Spirulina culture through light
with Nile red dye. Two drops of 1% (w/v) Nile red dye solution were
microscopy and scanning electron microscopy is shown in
added to 200 ␮L sample of the culture, vortexed and incubated for
Figs. 1 and 2. The Nile red staining of the S. subsalsa cells contain-
10 min at 55 ◦ C. Cells were taken on a glass slide and covered with
a coverslip. The stained cells were observed by fluorescent micro-
scope (Carl Zeiss Axio imager A1) at an excitation wavelength of
450–490 nm under 1000× magnification.

2.3. Estimation of cell dry weight

Cell growth was monitored gravimetrically and expressed in

terms of cell dry weight; the cell pellet obtained from a fixed culture
volume was dried in an oven until constant weight was achieved
to obtain the cellular biomass based on dry weight.

2.4. Extraction of polyhydroxyalkanoate (PHA)

Cells were harvested by centrifugation (8000 rpm, 10 min),

washed with distilled water and the biomass was suspended in
methanol overnight at 4 ◦ C for the removal of pigments. The pel-
let obtained after centrifugation was dried at 60 ◦ C and PHB was
extracted in hot chloroform. PHA was precipitated from the chlo-
roform solution into chilled methanol. The methanol–chloroform Fig. 1. Optical microscopic image of S. subsalsa (40×).
 A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260 257

Fig. 2. Scanning electron microscope image of S. subsalsa.

Fig. 5. PHA production by S. subsalsa under control and increased NaCl concentra-
tion.

Fig. 3. Nile red stained S. subsalsa cells containing PHA inclusions.

Fig. 6. FTIR spectra of PHA from S. subsalsa (SPS), compared with standard PHB
(Sigma).
ing PHA inclusions is shown in Fig. 3. PHA inclusions were seen as
bright (For interpretation of the references to colour in the text, the
3.2. Effect of increased NaCl concentration on PHA biosynthesis
reader is referred to the web version of the article.)orange intra-
cellular granules. The growth pattern of S. subsalsa in control and
Effect of NaCl concentration on PHA biosynthesis in Spirulina has
increased NaCl concentration is shown in Fig. 4. The culture entered
not been reported. The S. subsalsa culture incubated in increased
the stationary phase after 15 days.
NaCl concentration was compared with the S. subsalsa culture
incubated in normal condition (control) for PHA production 5.9%

Fig. 4. Growth behavior of S. subsalsa under control and increased NaCl concentra-
tion. Fig. 7. NMR spectra of the PHA produced by S. subsalsa and standard PHB (Sigma).
258 A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260

Table 1
Production of PHA by Spirulina subsalsa.

Spirulina subsalsa Cell dry weight (g/L) PHA content mg/g (cell dry weight) % Yield

Spirulina subsalsa (control) 2.2 129.8 5.9

Spirulina subsalsa (increased salinity) 1.97 147.75 7.45

PHA/CDW (w/w) as shown in Fig. 5. When the culture was incu-
bated with increased NaCl condition in the nitrogen free media,
the culture showed increased PHA synthesizing ability, which
amounted to a maximum of 7.45% PHA/CDW (w/w) (Table 1).
3.3. Chemical analysis of the PHA produced
The polymer obtained from S. subsalsa was characterized by
FTIR, NMR spectroscopy, TGA and DSC. Fig. 6 shows the FTIR spectra
of the PHA obtained from S. subsalsa and compared with the stan-
dard PHB from Sigma. FTIR analysis of the isolated polymer revealed
absorption bands at 1724 cm−1 , corresponding to the ester carbonyl
group.
Fig. 7 shows characteristic 500 MHz 1 H NMR spectra of the
PHA isolated from S. subsalsa grown in increased NaCl concen-
tration, which showed the following resonance signals: HC CH
at 5.30 ppm, CH2 COOH at 2.50 ppm, methylene groups ranging
from 1.25 to 1.57 ppm, and a terminal-CH3 at 0.9 ppm. The PHA iso-
lated from S. subsalsa was compared with the standard PHA from
Sigma, both show peaks that appear at almost identical chemical

Fig. 8. DSC spectra of second heating of PHA from S. subsalsa and standard PHB (Sigma). (a) DSC thermogram of S. subsalsa PHA and standard PHA (Sigma) showing melting
temperature peaks. (b) DSC thermogram showing glass transition temperature of S. subsalsa PHA and standard PHA (Sigma).
 A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
Fig. 9. TGA spectra of PHA from S. subsalsa and standard PHB.
shifts and integration values, indicating that they are very similar
in chemical composition, the DSC spectra recorded after the second
heating revealed that the glass transition temperature (Tg) value of
PHA from S. subsalsa was at 4.5 ◦ C and that of standard PHA is at
5.2 ◦ C. The melting temperature peaks observed for S. subsalsa PHA
is at 261 ◦ C and that of standard PHA is at 245 ◦ C, Fig. 8(a and b).
Thermal stability of PHA obtained was measured by TGA. The tem-
perature at 10% weight loss [Td(10%)] was studied. It was found
that the PHA from S. subsalsa had Td(10%) at 234 ◦ C as compared
to the Td(10%) of standard PHB from Sigma 260 ◦ C shown in Fig. 9.
Based on the characterization of the PHA produced by S. subsalsa
through FTIR, NMR, TGA, DSC and comparison with the standard
PHB (Sigma), it was observed that the PHA obtained from S. subsalsa
is having properties similar to that of the standard PHB (Sigma).
Usually the PHA in many chemo-organotrophic or photo-
heterotrophic microorganisms serves as an energy store, and the
unbalanced condition has effect on PHB accumulation [15]. This
distinctive behavior of cyanobacteria was ascribed to the lack
of a complete tricarboxylic cycle (TCA), which did not permit
dissimilation of acetyl-CoA [16]. Acetyl-CoA, derived from PHB,
might be used for biosynthetic purposes. The interrupted TCA
cycle in cyanobacteria serves predominantly to provide interme-
diates in biosynthetic pathways such as synthesis of amino acids,
carotenoids, chlorophyll, and PHB could be a specific carbon store.
PHB is a reduced compound it might act as a sink for an excess
of electrons. The positive effect of increased NaCl concentration
on PHA accumulation could be related to the enzyme activity
which is directly involved for the synthesis of the polyester. PHB
biosynthesis takes place by the condensation of two moles of
acetyl-CoA to acetoacetyl-CoA and the subsequent formation of
␤-hydroxybutyryl-CoA [17,18]. During imbalanced growth con-
ditions, citrate synthase is inhibited and levels of NADH and
acetyl-CoA increases. The concentration of free coenzyme A is
decreased, thus, the inhibition of 3-ketothiolase by coenzyme A
is released and the synthesis of PHB begins. Thus, increased NaCl
concentration helps in releasing the enzyme 3-ketothiolase which
is responsible for the synthesis of PHB and thereby increasing the
PHB synthesis in S. subsalsa when grown under increased salinity.
The production of PHA in S. subsalsa was enhanced by the addi-
tion of NaCl. It appears as though PHA accumulates in response to
259
different types of stressful conditions, such as those associated with
high salt concentration and under the condition of limiting nutri-
tional elements such as N, P, S, O or Mg in the presence of excess
carbon source [1]. One could speculate the possibility that the high
salinity conditions could allow for the redirection of carbon flux
towards PHA accumulation as a reserve compound.
The exact mechanism of biosynthesis of PHA in the presence
of increased NaCl concentration is still not completely understood.
A full interpretation of these results is not possible at this point
since, no data on the genes and enzymes involved in the synthesis
of PHA in Spirulina have been reported and requires further studies
in future to be carried out to determine the effect of NaCl on the
rate of accumulation of the PHA.
4. Conclusion
Cyanobacteria do have the potential to produce biopolymers
like PHA from CO2 as the sole carbon source, and the yield of
PHA could be increased by various means such as nutrient limiting
conditions, stress conditions, different PHA enhancing precursors,
recombinant strains, in vitro through enzymatic PHA synthase,
etc. The present study shows increased PHA accumulation in S.
subsalsa by twofold increased NaCl concentration in the growth
media. Characterization of the PHA produced was performed by
FTIR and NMR which confirms the chemical structure as compared
to the standard PHB (Sigma). Thermogravimetric analysis indicated
that the thermal stability of the polymer is more than 200 ◦ C. The
concentration of PHA produced is relatively lower in phototrophs
as compared to heterotrophic bacteria. Efficient metabolic/genetic
engineering is required to improve PHA yields in cyanobacte-
ria.
Due to their minimal nutrient requirement and ability to grow
even in wastewaters in the presence of CO2 and sunlight these pho-
totrophs (cyanobacteria) could be explored as an alternative source
for PHB production, as the biomass could be inexpensively con-
verted into biodegradable plastics by solar energy which can aid
in overall reduction of the production cost of biodegradable plas-
tics, which is the limiting factor for the replacement of synthetic
polymers by such biodegradable biopolymers.
260 A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260

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