Algal Research 33 (2018) 231–238

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Efficacy of Spirulina sp. polyhydroxyalkanoates extraction methods and T

influence on polymer properties and composition
⁎
Samantha Serra Costaa, , Andréa Lobo Mirandaa, Denilson de Jesus Assisb,
Carolina Oliveira Souzac, Michele Greque de Moraisd, Jorge Alberto Vieira Costad,
Janice Izabel Druzianc
a
Institute of Health Sciences, RENORBIO, Federal University of Bahia, Salvador, Bahia, Brazil
b
Department of Chemical Engineering, Polytechnic School, Federal University of Bahia, Salvador, Bahia, Brazil
c
Department of Bromatological Analysis, College of Pharmacy, Federal University of Bahia, Salvador, Bahia, Brazil
d
Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The objective of this study was to evaluate the efficacy of methods used to extract polyhydroxyalkanoates from
Cyanobacteria Spirulina sp. LEB-18 microalgae and to examine the influence of these methods on the purity, properties, and
Biopolymer composition of the polymers. Polyhydroxyalkanoates were characterized by Fourier transform infrared spec-
Polyhydroxyalkanoate troscopy (FTIR), molecular mass, degree of crystallinity, and monomer composition. The efficacy of the ex-
Microalgae
traction methods varied, with polyhydroxyalkanoate accumulation.
between 6.10 and 9.80%, and degrees of purity between 63.51 and 93.62%. Using sodium hypochlorite in the
initial stage of the extraction increased accumulation, while using methanol at the end of the process increased
the purity of the polymers. The molecular mass and crystallinity index of the polyhydroxyalkanoates varied,
showing that the extraction methods interfered with polymer properties. The composition of polyhydroxyalk-
anoates was also influenced by the extraction, with varying percentages of monomers identified. The copolymers
of the polyhydroxyalkanoates obtained are formed by the monomers 11-hydroxyhexadecanoate, in a higher
proportion, hydroxyheptanoate and hydroxytetradecanoate, demonstrating that Spirulina sp. LEB-18 is capable
of producing medium and long chain polymers. The detection of these monomer blocks in the poly-
hydroxyalkanoate structure of this microalga is an important scientific novelty because these monomer blocks
are constituents of new polymers. An indirect relationship (R2 = 0.8044) was observed between the percentage
of the 11-hydroxyhexadecanoate monomer and the degree of crystallinity of polyhydroxyalkanoates obtained by
the different methods. This suggests that obtaining the polymer with medium- and long-chain monomers con-
tributes to reduction of crystallinity.

1. Introduction these sole prokaryotes accumulate PHAs by oxygenic photosynthesis

Polyhydroxyalkanoates (PHAs) are polyesters that are synthesized
and accumulated intracellularly as granules by numerous microorgan-
isms [1,2]. The properties of PHAs, including thermoplastic processa-
bility, absolute resistance to water, and complete biodegradability,
suggest that PHAs can be a potential substitute for common plastics
[3–5]. Despite these attractive characteristics, the use of PHAs in food
packaging, biomedicine, pharmaceutical industry, and other applica-
tions is limited by high costs of production and extraction [2,6].
Cyanobacteria are a phylum of bacteria that can be used to obtain
PHAs at a lower cost. This is possible because cyanobacteria have
minimal nutrient requirements and possess photoautotrophic nature;
[7,8]. More than 100 cyanobacterial strains have been screened thus
far; approximately 70% of these strains contain PHAs at concentrations
ranging from 0.04 to 40% of dry cell weight under photoautotrophic
growth conditions [9].
The method used to extract PHAs also affects the costs of obtaining,
characteristics, and monomeric composition of the biopolymers, which,
in turn, impacts their industrial application. The properties and appli-
cation potential of these materials depend largely on monomer com-
position [6].
PHAs are comprised of monomers possessing a carbon backbone
from 4 to 16 carbon atoms long, and a broad range of functional groups
such as halogens, phenoxy, acetoxy, phenyl, cyano, and epoxy groups

⁎
Corresponding author.
E-mail address: manthasc@yahoo.com.br (S.S. Costa).

https://doi.org/10.1016/j.algal.2018.05.016
Received 13 December 2017; Received in revised form 17 May 2018; Accepted 18 May 2018
Available online 28 May 2018
2211-9264/ © 2018 Elsevier B.V. All rights reserved.
S.S. Costa et al.
[5,10]. The method of extraction is very important for monomeric
composition and can influence the properties of PHAs. Because it is a
product accumulated in the cellular cytoplasm, PHAs are extracted
from cells after production (culture) [1]. Extraction involves different
procedures that ensure proper removal of the biopolymer from the in-
terior of the cells. These include destabilization and/or cellular dis-
ruption, separation of the culture medium from the biomass, recovery
of the biopolymer, and purification [2,7]. The methods used for ex-
traction of PHAs include application of organic solvents; supercritical
fluids; biological digestion (enzymes); application of mechanical
methods, such as high-pressure homogenization and ultrasound; com-
bined mechanical and chemical methods; and studies of spontaneous
release of PHAs [5,6,11]. Extraction with organic solvents is employed
most because of ease of application, low degradation, and high purity of
the extracted product [12].
Because extraction methods are so important in obtaining PHAs, it is
essential to study how these methods and efficiency of processing affect
the molar mass, degree of crystallinity, monomer composition, and
other characteristics of the final product. These polymeric character-
istics are vital because they are directly related to the application and
degradation time of the polymer [6].
Thus, the objective of this work was to evaluate and compare the
efficacy of six different methods used to extract PHAs from the biomass
of Spirulina sp. LEB-18 microalgae; we then correlated the results with
the purity, composition, and properties of the obtained biopolymers.
2. Material and methods
2.1. Microorganism, culture medium, and cultivation conditions
In this study, we used Spirulina sp. LEB-18 isolated from the
Mangueira Lagoon (33°30′12″S, 53°08′58″W; Rio Grande, Brazil).
Zarrouk medium was used to maintain the inoculum of Spirulina sp.
LEB-18. The medium constituents were 16.8 g L−1 NaHCO3, 0.5 g L−1
K2HPO4, 2.5 g L−1 NaNO3, 1.0 g L−1 K2SO4, 1.0 g L−1 NaCl, 0.2 g L−1
MgSO4·7H2O, 0.04 g L−1 CaCl2, 0.01 g L−1 FeS04·7H2O, 0.08 g L−1
EDTA, and micronutrients [13].
Outdoor cultivation was conducted in raceway tanks with lengths,
breadths, and heights of 2.20 m, 0.90 m, and 0.35 m, respectively; these
tanks contained 240 L of Spirulina sp. LEB-18. The tank culture was
agitated by submersible pumps 24 h per day with water temperature
ranging from 37 to 26 °C. The volume of culture media was maintained
by periodic addition of potable water to compensate for evaporation
and maintain the volume at 240 L. Batch cultures were managed during
the month of August (winter). The cultures were maintained under
natural light, using a photoperiod of 12 h (light/dark), in the tank for
up to 30 days; the initial cell concentration was 0.2 g L−1. This period,
cell concentration was determined by optical density using a digital
spectrophotometer (BEL PHOTONICS UV-M51, Piracicaba – SP – Brazil)
at the wavelength of 670 nm. Before the experiments, a standard
growth curve of Spirulina sp. LEB-18 was generated and used to cor-
relate optical density with the dry-weight biomass (Cell concentration
g L−1 = 0.617 × Absorbance + 0.00125, R2 = 0.9916).
The biomass produced in the pilot scale was removed, separated by
centrifugation at 10000 rpm for 15 min (HITACHI CR22GIII, São Paulo
– SP – Brazil), frozen, lyophilized, vacuum-packed, and stored until
further testing.
2.2. Extraction methods
Six different methods were used to extract PHAs from the dry bio-
mass (Table 1). Each extraction method was tested using 3 g of dry
biomass. In the M 1 method, the biomass was washed with a solution of
4% sodium hypochlorite for 20 min at 45 °C and centrifuged. The su-
pernatant was discarded, and the precipitate was washed with distilled
water and re-centrifuged. The supernatant was discarded again, and
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Algal Research 33 (2018) 231–238
acetone was added for 2 h at 45 °C to precipitate the biopolymer. The
precipitate was dried in an oven at 35 °C for 48 h [11].
In the M 2 method, the biomass was washed with 4% sodium hy-
pochlorite for 20 min at 45 °C and then centrifuged. The supernatant
was discarded, and the polymer was extracted in hot chloroform for 3 h
at 80 °C, followed by precipitation from the chloroform solution into
chilled methanol. The methanol-chloroform mixture was decanted, and
the precipitated polymer was separated by centrifugation. Then, the
polymer was dissolved in chloroform and precipitated using evapora-
tion of the solvent according to Samrot et al. [14] with modifications.
In the M 3 method, the biomass was suspended in methanol over-
night at 4 °C to remove the pigments. The pellet obtained after cen-
trifugation was dried at 60 °C, and the polymers were extracted in hot
chloroform for 3 h at 80 °C. The PHAs was precipitated from the
chloroform solution into chilled methanol. The methanol-chloroform
mixture was decanted, and the precipitated polymer was separated by
centrifugation. Then, the polymer was dissolved in chloroform and
obtained via evaporation of the solvent [15].
Method M 4 was used according to Martins et al. [11] with mod-
ifications. First, the biomass was washed with 4% sodium hypochlorite
solution for 20 min at 45 °C and then centrifuged. The supernatant was
discarded, and the precipitate was washed with distilled water and re-
centrifuged. The supernatant was discarded again, and chloroform was
added for 3 h at 80 °C to precipitate the biopolymer; in this approach,
chloroform was used instead of the acetone used in Method M 1. The
precipitate was then dried in an oven at 35 °C for 48 h.
Method M 5 was used according to Penloglou et al. [16] with
modifications. The dried biomass was suspended in methanol overnight
at 4 °C to remove the pigments. The pellet, obtained after centrifuga-
tion, was dried at 60 °C, resuspended in distilled water, and incubated
in an ultrasonic bath for 30 min. Then, the sample was centrifuged, and
the supernatant was discarded. PHAs were extracted with hot chloro-
form for 3 h at 80 °C. Afterwards, the sample was filtered, and the
polymer was obtained via evaporation of the solvent.
The M 6 method was similar to the M 5 method, but excluded the
initial methanol-wash step. The dried biomass was resuspended in
distilled water, incubated in an ultrasonic bath for 30 min, and cen-
trifuged. PHAs were extracted with hot chloroform for 3 h at 80 °C. The
sample was then filtered, and the polymer was obtained via evaporation
of the solvent [16].
All extractions were performed in triplicate.
2.3. Accumulation of PHAs
The accumulation of PHAs was calculated gravimetrically, with
respect to the initial mass of dry biomass employed in each extraction,
according to Eq. (1).
mp × 100
Y=
mb (1)
where
Y is the accumulation of PHAs expressed as a percentage;
mp is the mass of PHAs obtained in grams;
mb is the mass of dry biomass used in the extraction and expressed
in grams.
2.4. Properties of PHAs
2.4.1. Fourier transform infrared spectroscopy (FTIR)
Samples of PHAs were qualitatively analyzed with Fourier trans-
form infrared spectroscopy (FTIR; PerkinElmer Model Spectrum 100)
between 4000 and 600 cm−1 using ATR accessory with a zinc selenide
crystal.
S.S. Costa et al. Algal Research 33 (2018) 231–238

Table 1
Methods used for the extraction of biopolymers.
Method Description Reference

M1 4% sodium hypochlorite wash (20 min/45 °C) Martins et al. [11]

Acetone wash (2 h/40 °C)
M2 4% sodium hypochlorite wash (20 min/45 °C) Samrot et al. [14] with modification
Extracted in hot chloroform (3 h/80 °C)
Precipitated from chloroform solution into chilled methanol
M3 Suspended in methanol (overnight/4 °C) Shrivastav et al. [15]
Extracted in hot chloroform (3 h/80 °C)
Precipitated from chloroform solution into chilled methanol
M4 4% sodium hypochlorite wash (20 min/45 °C) Martins et al. [11] with modifications
Extracted in hot chloroform (3 h/80 °C)
M5 Suspended in methanol (overnight/4 °C) Penloglou et al. [16] with modification
Ultrasound (30 min)
Extracted in hot chloroform (3 h/80 °C)
M6 Ultrasound (30 min) Penloglou et al. [16]
Extracted in hot chloroform (3 h/80 °C)

2.4.2. Molecular mass (Mm)
The Mms of PHAs were obtained via size-exclusion chromato-
graphy, using HPLC (PerkinElmer Series 200) equipped with an auto-
sampler and a refractive index (RI) detector (PerkinElmer), according to
Assis et al. [17].
For separation of PHAs, we used a Shodex KD 807 column
(30 cm × 78 mm × 5 mm) at 30 °C. The polymers were dissolved in
chloroform to a final concentration of 0.7 g L−1 and filtered through a
0.45-μm PTFE membrane before separation. The mobile phase was
chloroform at a flow rate of 1.0 mL min−1. A standard curve [Eq. (2)]
was created using low polydispersity polystyrene standards
(682–1.670.000 Da); (Polystyrene High Mw Standards Kit, Polymer
Standards Service, USA).
logMw = −0.8364 × Tr + 14.83 with R2 = 0.9917
2.4.3. X-ray diffraction (XRD)
X-ray powder diffraction analyses were performed using a SHIMA-
DZU (XRD-6000, USA) diffractometer operated at 40 kV and 30 mA
with graphite-filtered CuKa (λ = 1.5433°A) radiation. The data were
acquired on a 2θ scale from 5° to 60°. XRD was used to determine the
degree of crystallinity of PHAs samples. The percentage of crystallinity
was calculated from the diffracted intensity, which was measured by
XRD according to the Vonk method [18,20].
2.4.4. Monomeric composition of PHAs
Identification and quantification of the constituent monomers of
PHAs were performed using gas chromatography/mass spectrometry
(GC–MS) (Clarus 500 PerkinElmer) with TurboMass software v. 4.5.0
and NIST 98 library. PHAs (~0.04 g) were subjected to methanolysis
using methods described in the literature [21] with some modifications
[22]. All PHAs samples were dissolved in 2 mL of acidified methanol
containing 3% (v/v) H2SO4 and 1 mL of chloroform in screw-capped
test tubes. The samples were then incubated at 100 °C for 60 min. After
cooling to ambient temperature, 1 mL distilled water was added and the
sample was shaken for 10 min, after which the two phases were per-
mitted to separate [22].
The organic phase was separated after splitless injection into a ca-
pillary column DB-1 (30 m × 0.25 mm × 0.25 mm). Helium (flow rate
1.0 mL min−1) was used as the carrier gas. The temperatures of the
injector and detector were 250 and 240 °C, respectively. The mass
spectrometer was programmed to scan between 50 and 550 m/z with a
temperature program of 80–200 °C (20 °C min−1). The mass spectra
were compared with the spectra from 98 NIST library, and hydro-
xyalkanoate monomers were quantified by area normalization.
2.5. Statistical analysis
(2) The responses were assessed using an analysis of variance followed
by Tukey's test at a 95.0% confidence level. Experimental results were
analyzed using STATISTICA 7.0 software.
3. Results and discussion
3.1. Accumulation of PHAs
The culture had a cell concentration of 1.02 g L−1 at the end of the
30 days, yielding approximately 245 g of dry biomass. The growth
curve of Spirulina sp. LEB-18 under the same culture conditions is
shown in Jesus et al. [23]. Accumulation of PHAs (Table 2) varied from
6.10 to 9.80% and differed significantly (p < 0.05) between the
methods used for extraction. These results indicate that the methods
differ with respect to the levels of efficacy, which may be related to the
purity of the obtained biopolymers. The M 4 method, which used so-
dium hypochlorite, showed higher PHAs accumulation (Table 2) com-
pared with the M 6 method in which the biomass was subjected to
ultrasound. Sodium hypochlorite and ultrasound are used to break the
cell membrane in microorganisms, thereby enabling the extraction of
PHAs that are accumulated intracellularly.
Ultrasound creates a mechanical disturbance in which ultrasonic

Table 2
Accumulation and purity of Spirulina sp. LEB-18 PHAs according to the extraction method.
Extraction method Accumulation (%, w/w dry biomass) Purity (%) Accumulation PHAs pure (%)

a
M 1 Sodium hypochlorite + acetone 8.06 ± 0.20 89.35 7.20 ± 0.20a
M 2 Sodium hypochlorite + chloroform + methanol 8.70 ± 0.30b 93.62 8.14 ± 0.30b
M 3 Methanol + chloroform + methanol 6.10 ± 0.30c 85.17 5.20 ± 0.30c
M 4 Sodium hypochlorite + chloroform 9.80 ± 0.40d 79.84 7.82 ± 0.40d
M 5 Methanol + ultrasound + chloroform 6.70 ± 0.40c 84.24 5.64 ± 0.40c
M 6 Ultrasound + chloroform 8.91 ± 0.20b 63.51 5.66 ± 0.20c

Means ± standard deviations, n = 3. The equal letters in the same column indicate no significant differences between experiments, assessed at 95% confidence level
(p < 0.05).

233
S.S. Costa et al.
waves are transferred and dissipated in a cellular suspension; this forms
a type of field in which there occurs an increase of mass and consequent
transfer of heat to the liquid medium. This produces a velocity gradient
and creates a force capable of destabilizing the cell wall [24]. Thus,
with the use of ultrasound, there is greater facility for solvent action
and, consequently, the recovery of the obtained polymer is improved
[16].
When this physical method is used to disrupt the cell wall of mi-
croorganisms, a solvent (chemical method) is employed later to extract
the polymer. This was used in the M 6 method, where after the use of
ultrasound, chloroform was used for biopolymer extraction (Table 1).
The M 6 method showed a PHAs accumulation of 8.91%, approximately
9% lower than the accumulation obtained using sodium hypochlorite in
the initial step (9.80% of accumulation, chemical method M 4)
(Table 2). These data indicate that the chemical method was more ef-
ficient in destabilizing and rupturing the cell wall of the microalgae
biomass than was the physical method (ultrasound).
The chemical method consisted of combining the biomass with a
solvent at a high temperature (60 to 80 °C). In this case, permeability of
the cell membrane is modified by the solvent and solubilization of the
polymer, forming a suspension of cellular debris in the solution of the
polymer and solvent [25]. The solid polymer in contact with the solvent
tends to swell, forming a gel; this is due to diffusion of the solvent
molecules into the polymer mass in the free volume between the
polymer chains [24].
When hypochlorite is used in the initial stage of extraction, an in-
verse process occurs because hypochlorite breaks the cell membrane
and dissolves the non-PHAs cellular material, leaving the insoluble
polymer precipitating in the solution. With the subsequent use of a
solvent in which the polymer is not soluble, extraction of the same
tends to occur with higher purity [14]. Numerous solvents (such as
chloroform, acetone, methyl isobutyl ketone, methylene chloride, pro-
pylene carbonate, ethyl acetate, and isoamyl alcohol) can be used to
extract PHAs, with chloroform being the most used in the extraction
stage [20,22].
Sharma et al. [9] used sodium hypochlorite followed by hot
chloroform (method M 4, Table 1) to extract PHAs from the cyano-
bacteria Nostoc muscorum biomass; they obtained PHAs accumulation of
up to 47.4% by adding glucose and acetate to the microalgae culture
medium. Jau et al. [26] also used sodium hypochlorite at the initial
PHAs extraction stage of Spirulina sp. LEB-18 biomass and obtained a
PHAs accumulation of 10.1%, similar to that obtained in our study.
Penloglou et al. [16] used ultrasound in the initial stage to extract PHAs
from the biomass of the bacterium Alcaligenes latus grown in a con-
tinuous process; they obtained PHAs accumulation between 4 and 12%,
depending on the concentration of the carbon source used in the
medium. This shows that only the extraction method interferes with
PHAs accumulation.
In this study, in five (M 2, M 3, M 4, M 5, and M 6) of the six
methods evaluated, chloroform was used to extract the PHAs; only in
the M 1 method, acetone was used as extraction solvent (Table 1).
Comparing the efficacy of these two solvents (Table 2) showed that
with chloroform, the accumulation of PHAs (9.80% of accumulation)
was approximately 18% higher than that obtained with acetone (8.06%
of accumulation). However, acetone is widely used for extracting PHAs
from microalgal biomass because it is less costly and less toxic than
chloroform [27,28], which may justify its use. Martins et al. [11] used
acetone as PHAs extraction solvent for Spirulina sp. LEB-18 biomass and
reported PHAs accumulation between 7.64 and 44.19%, higher than
that obtained in this study. The highest percentages of PHAs accumu-
lation, reported by Martins et al. [11], are likely due to variations in the
concentration and source of carbon used in the culture medium, which
favored higher production of PHAs by the microalgae.
In the M 2, M 3, and M 5 methods, we washed the biomass with
methanol (Table 1). This solvent was used at the beginning of the ex-
traction process (M 3 and M 5) to remove the pigments present in the
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Algal Research 33 (2018) 231–238
biomass, which may interfere with the extraction of the biopolymers.
This step is widely used in extracting PHAs from a microalgal biomass
because these microorganisms possess high levels of pigments; these
pigments can be extracted together with PHAs and interfere with ex-
traction efficiency [12,29]. When methanol is employed at the end of
the extraction process (M 2 and M 3, Table 1), it is used to precipitate
the polymer, which is in solution with the extraction solvent. This step
is important because it can increase the purity of the recovered PHAs.
When used for this purpose, methanol can be replaced by a non-solvent,
such as mixtures of alcohols and water, or by a physical method of
separation, such as filtration or centrifugation; these approaches were
used in the methods M 1, M 4, M 5, and M 6 (Table 1). A non-solvent is
defined as a substance that cannot dissolve the biopolymer via pre-
cipitating it by induction and/or by weakening the solvent's dilution
power [30].
Thus, methods using methanol produced lower levels of extracted
PHAs than methods that did not use methanol. This was because using
methanol results in smaller amounts of interfering compounds and,
consequently, greater purity of extracted PHAs. This was shown by
comparing the M 4 and M 2 methods, in which PHAs accumulation
increased from 9.80 to 8.70%, respectively, and by M 6 and M 5
methods, where PHAs accumulation increased from 8.91 to 6.70%,
respectively, when methanol was used at the end of the extraction
process (Table 2). Shrivastav et al. [15] used methanol at the beginning
and end of PHAs extraction from the cyanobacterium Spirulina subsalsa,
cultivated in ASNIII medium, and obtained a PHAs accumulation of
7.45% after 10 days of cultivation. Kavitha et al. [6] used washing with
sodium hypochlorite, followed by extraction with hot chloroform and
precipitation in cold methanol (M 2 method), to extract PHAs from the
biomass of Botryococcus braunii microalgae; they obtained an accumu-
lation of 20.4% using a temperature of 40 °C and culture medium that
incorporated 60% sewage.
3.2. Properties of PHAs
3.2.1. FTIR
Fig. 1 shows the FTIR spectra of PHAs samples obtained using each
method and shows a similarity between the samples overall. The
transmittance bands located at 1735–1745 cm−1 were attributed to the
stretching vibration of the C]O group (ester carbonyl) in the PHAs
polyester. Accompanying bands of the CeOeC groups appear in the
spectral region from 1285 to 1310 cm−1. The transmittance region from
2800 to 3100 cm−1 corresponds to stretching vibration of the CeH
Fig. 1. FTIR spectra of PHAs extracted from Spirulina sp. LEB-18 using different
extraction methods.
S.S. Costa et al.
Table 3
Weighted average molecular mass and crystallinity index of PHAs obtained by
different extraction methods.
Methods Mm (kDa) Crystallinity (%)
M 1 61.07 ± 0.03a 50.47
M 2 58.76 ± 0.09b 50.15
M 3 58.76 ± 0.04b 51.20
M 4 58.76 ± 0.03b 51.32
M 5 62.26 ± 0.03c 51.18
M 6 62.26 ± 0.05c 51.65
Means ± standard deviations, n = 3. The equal letters in the same column
indicate that there was no significant difference between the experiments, as-
sessed at 95% confidence level (p < 0.05).
bonds of methyl (CH3) and methylene (CH2) groups. The band at
700 cm−1 corresponds to the ReCeO group [31,33]. These functional
groups are characteristic of the chemical structure of PHAs, confirming
that all the extraction methods resulted in PHAs as major compounds.
However, different levels of band intensity were observed among the
samples obtained by the different methods; this suggests different levels
of pure PHAs among the samples and was confirmed in the analysis of
monomeric composition.
Shrivastav et al. [15] examined the FTIR spectra of biopolymer
samples obtained from Spirulina subsalsa biomass and reported bands at
1724 cm−1, corresponding to the ester carbonyl group. Kavitha et al.
[6] analyzed the FTIR spectra of polyhydroxybutyrate extracted from
the biomass of Botryococcus braunii microalgae and identified absorp-
tion bands at: 2933 cm−1, corresponding to the eCH3 group; at
1652 cm−1, corresponding to the ester-carboxylic group; and at
1232 cm−1, which is characteristic of the asymmetric and symmetrical
stretching vibrations of the CeOeC groups; these data indicate that the
biopolymer was obtained.
3.2.2. Molecular mass
Table 3 shows the average molecular masses of PHAs obtained by
the different extraction methods. The molecular mass ranged from
58.76 to 62.26 kDa with significant differences (p < 0.05) between the
samples, confirming that the extraction method affected the molecular
weight of PHAs. The extraction methods M 2, M 3, and M 4 produced
PHAs with lower molecular mass (58.76 kDa) and did not cause sig-
nificant difference between the samples (p > 0.05). In contrast, the M
1, M 5, and M 6 methods produced PHAs with molecular mass up to 5%
higher than that of samples extracted using other methods; the M 1, M
5, and M 6 methods generated significant differences between the
samples (p < 0.05) (Table 3).
The M 5 and M 6 methods, which used the ultrasound at the initial
stage (Table 1), produced PHAs with a molecular mass (62.26 kDa) 5%
higher than that produced with methods that used sodium hypochlorite
(M 2, M 3, and M 4); this shows that the more aggressive chemical
method produced polymers with lower molecular mass (Table 3). This
results from combining the solvent with a high temperature, which can
cause the breakdown of the polymer and reduce the molecular mass of
the polymer chain [24].
According to Kulkarni et al. [2], thermoplastic polymers are char-
acterized by covalent bonds between molecules, which can be disrupted
by the insertion of energy. Therefore, when polymers are heated, if the
thermal energy imposed on the polymer is higher than the energy of the
chemical bonds, some of these bonds may rupture; this causes a de-
crease in the molecular mass of the polymer [34].
We evaluated the extraction solvents used in each method. Using
acetone produced a polymer with a higher molecular mass, showing
that acetone was less aggressive in the extraction of the polymer
(Table 3). This is evidenced by comparing the M 1 method (acetone)
with the M 4 method (chloroform). In the M 4 extraction, a 4% re-
duction in the molecular mass of PHAs occurred with the use of
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Algal Research 33 (2018) 231–238
chloroform combined with a higher temperature (80 °C) (Table 3). The
PHAs extracted using the M 4 and M 5 methods had higher molecular
weights than those obtained by the other methods. This was attributed
to not using a chemical method at the beginning of the extraction
process, which is an important factor in reducing the molecular mass of
the polymer [6,24]. Even if the chemical structure of the biopolymer is
the same, different molar masses can significantly change the properties
of PHAs, such as physical, mechanical, thermal, rheological, processing,
and other properties. This is because these properties are dependent on
the size of the polymer chain of the biopolymer.
In this context, our results indicate that the average molecular mass
of PHAs can be influenced not only by the composition of the culture
medium and production conditions [2,22,35], but also by the method
used in the extraction of the polymer. This difference in behavior, ob-
served for polymers obtained in this study, was likely caused a struc-
tural rearrangement of the polymer during extraction. Because of in-
dustrial competition, the ability to control the molecular mass of the
biopolymer during production and extraction, and understanding how
these processes influence the final properties of the polymer, is ex-
tremely important.
Kulkarni et al. [2] reported a molecular mass of 208 KDa for PHAs
extracted with chloroform from the biomass of the cyanobacterium
Halomonas campisalis after 42 days of cultivation in added medium of
maltose and calcium chloride; this value is three times higher than that
found in our study. Campos et al. [22] obtained molecular masses be-
tween 510 and 745 kDa for PHAs extracted with chloroform from the
Cupriavidus necator bacteria cultivated with varying concentrations of
carbon and nitrogen; these values are also above those obtained in this
study. Searching the literature did not show studies reporting on the
molecular mass of PHAs extracted from the biomass of the Spirulina sp.
Different microorganisms produce PHAs with different molecular
masses. Culture conditions, such as the type and concentration of the
substrate, nutrient availability, pH, temperature of the culture medium,
and extraction methods, may also interfere with the molecular weight
of the polymer [2,30]. The low molecular weights of the PHAs pro-
duced by Spirulina sp. LEB-18 in this study were likely due to differ-
ences in the metabolic and polymerization processes involved in
polymer synthesis; these processes may differ compared to those of
other microorganisms, such as bacteria. The cyclic nature of PHAs
metabolism allows the production and degradation of the polymer to
occur simultaneously, which may directly affect the molecular mass of
the polymer. The level and time of expression of the PHAs synthase
enzyme, which varies with the type and species of microorganism, also
impacts the molecular mass of the polymer [30]. The molecular mass of
polymers is a property fundamental for their industrial application.
Therefore, all these factors, which directly affect the length of the
polymer chain, need detailed studies in order to develop models that
can predict the molecular mass of these polymers [8,30].
3.2.3. X-ray diffraction
The XRD profile (Fig. 2) showed peak diffraction at 2θ, which is
typical of semi-crystalline polyesters, at approximately 13.0°, 16.40°,
21.77°, and 24.96° for all samples of PHAs, indicating a crystalline
phase in the compositions [20]. A discreet variation (50.15 to 51.65%)
was observed for PHAs samples extracted using the different methods
(Table 3). The M 1 and M 2 methods, which used sodium hypochlorite
at the initial extraction stage (Table 1), showed the lowest crystallinity
degree of the polymers despite having the lowest molecular mass
(Table 3). The degree of crystallinity is directly related to processability
and industrial applications of PHAs. According to Laycock et al. [29],
crystallinity equal to or above 50% is considered high and detrimental
for commercial and industrial purposes. A low degree of crystallinity
increases the number of possible industrial applications of PHAs, im-
proving their processing characteristics.
Campos et al. [22] found a degree of crystallinity between 52.23
and 66.12% for PHAs obtained from Cupriavidus necator bacteria
S.S. Costa et al.
Fig. 2. X-ray diffractograms of PHAs obtained from Spirulina sp. LEB-18 by
different extraction methods.
cultivated in a medium with varying concentrations of carbon and ni-
trogen; this shows the influence of the medium on the degree of crys-
tallinity of the polymer. Ribeiro et al. [20] obtained PHAs from B. ce-
pacia bacteria grown on different carbon sources and reported a discrete
variation in the degree of crystallinity (49.70 to 48.10%) of the ob-
tained polymers; this shows that these microorganisms can biosynthe-
size PHAs with the same degree of crystallinity, regardless of the
composition of the culture medium.
3.2.4. Monomer composition of PHAs
In addition to the molecular mass and degree of crystallinity,
monomer composition of PHAs polymers was also influenced by the
extraction method used. The produced samples of PHAs were composed
of three building blocks of 7–16 carbon atoms. The formed copolymers
consisted mainly of the monomer block of 11-hydroxyhexadecanoate
(16 carbon atoms, 55.54 to 81.05%), identified by the retention time of
8.23 min using the NIST library (Table 4). However, the building blocks
of the hydroxyheptanoate monomers (7 carbon atoms, 4.65 to 9.95%)
and hydroxytetradecanoate (14 carbon atoms, 2.80 to 7.23%) are pre-
sent in lesser amounts, the formation of a heteropolymer of PHAs in the
biomass of the microalga Spirulina sp. The characterization of PHAs by
GC–MS indicated that this cyanobacterium can produce PHAs con-
sisting of copolymers with long- and medium-chain building blocks.
The biosynthesis of PHAs in microalgae is derived from metabolism of
fatty acids, in which intermediates can be eliminated and incorporated
into a polyester, forming longer chain structures. Thus, in microalgae, it
is common to obtain a heteropolymer consisting of monomers ranging
from 6 to 14 carbon atoms [36]. Detection of these monomer blocks in
the PHAs structure of Spirulina sp. is a genuine scientific novelty be-
cause they are constituents of completely new polymers.
Our results indicate that the PHAs obtained by the different
Table 4
GC–MS analysis of the composition (% area/mass) of PHAs obtained from Spirulina sp. LEB-18 by different extraction methods.
Retention time (min) Identification NIST Methods
M1
5.40 Methyl hydroxyheptanoate 4.93 ± 0.03a
6.48 Methyl hydroxytetradecanoate 5.08 ± 0.03a
8.23 11-Hydroxyhexadecanoate 79.34 ± 0.04a
Other peaks Unidentified 8.65 ± 0.03a
Means ± standard deviations, n = 3. The equal letters in the same column indicate that there was no significant difference between the experiments, assessed at
95% confidence level (p < 0.05).
Algal Research 33 (2018) 231–238
extraction methods showed different percentages of the monomers,
generating polymers with different monomeric compositions (Table 4),
molecular masses, and degrees of crystallinity; this caused differences
in the properties of the polymers and can affect their industrial appli-
cation. Our results show that the chemical composition of PHAs syn-
thesized by microorganisms depends not only on the carbon source
used during PHAs accumulation [22,37], but also on the method used
for extraction of the polymer.
Our results show that the methods employing sodium hypochlorite
in the initial step of polymer extraction (M 1 and M 2, Table 1) can
potentially improve the adaptability of the polymers with building
blocks of 14 or 16 units in their chains (79.42% - M 1, 85.24% - M 2,
Table 4); this confirms that the extraction step influenced the re-
arrangement and formation of the polymer chains.
With respect to the properties of PHAs, an indirect relationship
(R2 = 0.8044) was observed between the percentage of the 11-hydro-
xyhexadecanoate monomer and degree of crystallinity obtained for
PHAs samples extracted via different methods. These results indicate
that the complex organization of building blocks with medium and long
chains may have prevented precise formation and distribution of crys-
talline spheres in the polymer material, contributing to the reduction in
their crystallinity [30,38]. According to Ribeiro et al. [20], the modulus
and tensile strength of these copolymers decreases with increasing
monomer concentration as the greatest number of carbon atoms in the
chain, and the elongation to break increases satisfactorily.
By evaluating the monomeric composition of the polymers and
calculating the purity of the PHAs (Table 2), we found that the per-
centage of non-hydroxyalkanoate compounds, which were extracted
together with the polymer, varied (Table 4). Fig. 3 shows that the
percentages of non-hydroxyalkanoate compounds, identified in the
PHAs samples, varied from 6.38% for M 2 to 36.49% for M 6; this
demonstrates that the efficiencies of the extraction methods varied with
respect to the purity of the obtained biopolymers. We analyzed the
percentage of pure PHAs obtained by each method and correlated it
with PHAs accumulation (Table 2). Our analysis indicates that the M 2
method was the most efficient in extracting polymers with higher purity
(93.62%), generating an accumulation of pure PHAs of 8.14%; the M 3,
M 5, and M 6 methods were the least efficient, showing an accumula-
tion of pure PHAs of 5.20, 5.64, and 5.66%, respectively.
The methods in which the biomass was washed with sodium hy-
pochlorite (M 1, M 2, and M 4) were more efficient in the extraction of
biopolymers, confirming that this treatment was more effective for
rupturing cell membranes of cyanobacteria and allowing for greater
extraction of PHAs. Using methanol during biopolymer precipitation
stage (M 2) was important for obtaining a higher purity polymer with a
lower percentage of non-hydroxyalkanoate compounds; this step was
also important in extraction of PHAs.
4. Conclusion
This study demonstrated that the methods used for extracting PHAs
from the cyanobacterium Spirulina sp. LEB-18 show different effi-
ciencies in the extraction of the polymers. The use of sodium
M2 M3 M4 M5 M6
8.38 ± 0.02b 9.95 ± 0.05c 9.80 ± 0.03d 4.65 ± 0.04e 5.17 ± 0.02f
4.19 ± 0.03b 7.23 ± 0.04c 6.34 ± 0.06d 4.05 ± 0.02e 2.80 ± 0.03f
81.05 ± 0.03b 67.99 ± 0.05c 63.7 ± 0.03d 75.54 ± 0.05e 55.54 ± 0.03f
6.38 ± 0.03b 14.83 ± 0.03c 20.16 ± 0.03d 15.76 ± 0.05e 36.49 ± 0.03f
236
S.S. Costa et al.
Fig. 3. Quantification of hydroxyalkanoates and non-hydroxyalkanoate compounds obtained from Spirulina sp. LEB-18 via different extraction methods.
hypochlorite in the initial extraction stage increases polymer accumu-
lation, while the use of methanol at the end of the process is important
for obtaining higher purity PHAs. The extraction methods significantly
influenced (p < 0.05) the molecular mass, degree of crystallinity, and
monomeric composition of PHAs, showing that the extraction method is
critical in obtaining polymers with desired characteristics for industrial
applications. The PHAs extracted from the Spirulina sp. were composed
largely of the 11-hydroxyhexadecanoate monomers and hydro-
xytetradecanoate, which is a scientific novelty because these building
blocks are constituents of completely new polymers.
Acknowledgements
The authors acknowledge CNPq (National Council of Technological
and Scientific Development) for the financing of the productivity pro-
ject n° 311392/2016-4, FAPESB (Foundation for Research Support of
the Country of Bahia) by the doctorate scholarship and MCTI (Ministry
of Science Technology and Innovation).
Conflict of interest
The authors declare no conflict of interest.
Statement of informed consent, human / animal rights
No conflicts, informed consent, human or animal rights applicable.
Declaration of authors' agreement
All authors agree to the authorship and submission of the manu-
script to Algal research for peer review.
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