Biocatalysis and Agricultural Biotechnology 22 (2019) 101358

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Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

Extraction and characterization of polyhydroxyalkanoates from marine

green alga and cyanobacteria
*
K. Roja, D. Ruben Sudhakar, Susaimanickam Anto, Thangavel Mathimani
Department of Energy and Environment, National Institute of Technology, Tiruchirappalli, 620015, Tamil Nadu, India

ARTICLEINFO
ABSTRACT
Keywords:
Microalgae The present study focusses on the extraction and characterization of polyhydroxyalkanoates from marine
Cyanobacteria algal species by solvent extraction method. The following strains (i) Chlorella sp., (ii) Oscillatoria salina, (iii)
Bioplastic Lep- tolyngbya valderiana and (iv) Synechococcus elongatus were used in the present research work. Algal
Polyhydroxyalkanoates strains were scaled up in ASN III medium and preliminary bioprospecting study was conducted to evaluate
Solvent extraction the growth profile of all algal strains in terms of dry cell weight. Of the strains studied, the higher
Characterization
growth was observed with Leptolyngbya valderiana and Chlorella sp. at about 2.75 g/L whereas Oscillatoria
salina and Synechococcus elongatus showed low dry cell weight of 0.99 g/L and 0.32 g/L respectively. In
addition, extraction of poly- hydroxyalkanoates from the algal strains were carried out and characterized.
Thermo Gravimetric Analysis and Differential Scanning Calorimetry studies were conducted to determine
the thermal stability and thermal properties of the synthesized PHA respectively. All the PHAs were
thermally stable below 260 � C. PHA obtained from Leptolyngbya valderiana was found to be more
thermally stable compared to the PHA obtained from other algal species.

1. Introduction
3-hydroxyvalerate, 3-hydroxypropionate, 3-hydroxyhexanoate,
In the environment, non-biodegradable plastic waste accumulation 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecanoate, and
is estimated between 4.8 and 12.7 million tons per year, which 3-hydroxytetradecanoate. PHAs can be used as packaging components,
biodegradable printing inks, coatings and laminations.
creates major issues to the marine ecosystems (Lo€hr et al., 2017).
Additionally, these can be used in waxes, binders and adhesives as
This is due to the use of petrochemicals (non-renewable) as key
the PHA structures include different grades of thermoplastic
sources for plastic production currently (Sabapathy et al., 2019).
elastomers and rigid thermoplastics.
Hence, there is a search for degradable sources of plastics or
alternatives to non-biodegradable plastics, to reduce the Polyhydroxybutyrate is a type of PHAs. PHB is produced by
accumulation of plastic in the ecosystem. At this juncture, numerous microorganisms from acetyl-CoA through a series of
polyhydroxyalkanoates (PHA), a biopolyesters, an energy and enzy-
carbon source, can be synthesized by several microorganisms. PHAs matic reactions using three enzymes. β-ketothiolase (phaA) is the
have similar characteristics to the plastics made from first
petrochemical origin (Martins et al., 2014). PHAs extracted from key enzyme that converts acetyl-CoA in to acetoacetyl-CoA
microorganisms are envi- ronmentally benign and utilized in molecule; the second enzyme acetoacetyl-CoA reductase (phaB)
various medical and food sectors (Cassuriaga et al., 2018). Hydroxy reduces the acetoacetyl-CoA to 3-hydroxybutyrlCoA and the final
acid (HA) monomeric units linked together by ester bonds is the enzyme PHB po- lymerase (phaC) catalyses the polymerization of 3-
structural chemistry of PHA and are cate- gorized based on the hydroxybutyrlCoA to PHB molecule. In order to keep the PHB
number of carbons in the side-chains. There are short chain length polymerase covalently attached, PHB polymers tend to form
(scl) type which has less than 5 carbons, medium chain amphipathic granules within the cell. Among the microbes used for
length (mcl) type having 5–14 carbon atoms and long chain length PHA extraction, algae have gained substantial interest as they are
sunlight driven cell factories that convert CO2 into various valuable
(lcl)
type with more than 14 carbon atoms (Kunasundari and Sudesh, bio-products with the O2 evolution. Also, the route of bioplastic
production from algal source can be via the polymers from direct
2011). Common PHA monomers are 4-hydroxybutyrate, 3-
biomass or from its secondary metabolites (Abdo and Ali, 2019).
hydroxybutyrate,

* Corresponding author.
E-mail address: [email protected] (T. Mathimani).

https://doi.org/10.1016/j.bcab.2019.101358
Received 12 August 2019; Received in revised form 17 September 2019; Accepted 23 September 2019
Available online 24 September 2019
1878-8181/© 2019 Elsevier Ltd. All rights reserved.
K. Roja et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101358

Table 1
PHA content (%) in various algae.
Algae PHA content References
(%)
Synechococcus subsalsus 16 Costa et al., (2018a)
Spirulina sp. LEB-18 12 Costa et al., (2018a)
Phaeoda 10.6 Hempel et al., (2011)
ctylum
tricorn
utum
Chlamyd Dete Chaogang et al., (2010)
omonas ctabl
reinhar e
dtii quan
tities
Synechocystis PCC6803 4.1–26 (Khetkorn et al., 2016; Panda and
Mallick, 2007; Wu et al., 2001)
Synechococcus MA19 55 Nishioka et al., (2001)
Arthrospira subsalsa 14.7 Shrivastav et al., (2010)
8.7–22
(Panda et al., 2005;
Sharma and Mallick, 2005)
Phormidium sp. TISTR 8462 14.8 Kaewbai-ngam et al., (2016)
Oscillatoria 15.7 Kaewbai-ngam et al., (2016)
jasorvensis TISTR
8980 25.2 Kaewbai-ngam et al., (2016)
Calothrix
scytonemicola
TISTR 8095
Anabaena sp. 2.3 Lama et al., studied, PHB is the prevalent bioplastic in the
(1996)
Aulosira fertilissima 10 Samantaray and
prokaryotic cyanobacteria (Sun- daramoorthy et
Mallick (2012) al., 2013). In fact, the PHAs accumulation
Calothrix sp. 6.4 Bhati et al., in
(2010)
Scytonema sp. 7.4 Bhati et al.,
(2010)

Fig. 1. Time course analysis of DCW (a) Chlorella

Algae majorly are of two types prokaryotic and sp. (b) O. salina.
eukaryotic based on well-defined nucleus.
Prokaryotic algae are known as cyanobacteria or
blue green algae which are capable of
accumulating poly- hydroxyalkanoates under
photoautotrophic as well as heterotrophic
conditions (Table 1).
In cyanobacteria, the most dominant
intracellular storage compound identified is PHB
and further, of the 150 polyhydroxyalkanoids

2
K. Roja et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101358
izable sources are way better in i) recyclable
molecules: property ii) biodegradability iii) biocom-
these are patibility iv) plasticizing capacity v) usability in
made from multiple sectors such as medical, agricultural,
triglycerides industrial, food packing and storage materials etc
and lactic than the conventional petroleum based plastics
acid. PHB in (Abdo and Ali, 2019; Das et al., 2018). One of
algal cell the key challenges in PHA production from
principally algal source is the process economization.
acts as an Algal based bioplastics have innumerable
energy benefits compared to the present era of
storage recalcitrant pe- troleum based plastics in terms
molecule of degradation. Concurrently, the need of
wherein its bioplastics from microbial origin has been
use comes expanded in various fields for which the
into present productivity of biopolymers does not
existence suffice to achieve the commercial production
Fig. 2. Time
course when algal process. Moreover, bioprospecting of suitable
analysis of cells are in algal strain having high PHB content is of
DCW (a) L. shortage of much significance
valderiana macronu-
(b) S.
elongatus. trients,
thereby
deriving
cyanobacter energy for
ia were first its survival.
observed PHB are
under generally
ambient ob- tained
(photoautotr in low
ophic) quantity
conditions through
and it was carbon
increased fixation or
under carbon
organic assimilation
carbon process and
supplements can be
such as significantly
glucose, raised under
acetate, nutrient
citrate, limited
xylose, conditions
arabinose resulting in
etc. excessive
(Cassuriaga storage of
et al., 2018; carbon
Singh et al., compounds
2017). (Das et al.,
Cyanobacte 2018). The
rial reported
bioplastics microalgal
can be strains for
made using PHB
biopolymer production
s derived in the recent
from two times are C.
ways. They reinhardtii,
are (i) C. vulgaris,
biopolymer C. fusca
s from (Cassuriaga
living et al., 2018;
organism: Kato, 2019;
from Mathiot et
cellulose, al., 2019).
soy protein The PHB
and starch, production
(ii) polymer- from algal

3
 Fig. 3. FTIR Spectra of PHA (a) Chlorella sp. (b) O. salina.

and to the fact, only few microalgal strains have been reported for uchirappalli, Tamil Nadu for the present study. Further, algal
PHB production, thus claiming to search for highly productive sample
cyanobacterial and microalgal strains from diverse sources. Hence,
the present study initially focusses on selection of suitable algal
candidate based on its growth profile. Then the further study aims
at the extraction and char- acterization of polyhydroxyalkanoates
from three cyanobacterial spe- cies and one green algal species to
evaluate their potential for bioplastic.

2. Materials and methods

2.1. Strains and culture conditions

Three cyanobacteria were obtained from National Repository for

Microalgae and Cyanobacteria (NRMC), Bharathidasan University, Tir-
was isolated from the saltpan of Kanyakumari, Tamil Nadu and it
was purified for further studies. The following strains cultures
were grown in ASN III medium in a temperature controlled culture
room at 25 2 � C. All the cultures were illuminated by cool �
fluorescent lamp at 1500 lux with 14:10 L/D photoperiod.
Cultures used in the present study are, a. Synechococcus elongates
(S. elongates) BDU 30312, b. Leptolyngbya val- deriana (L.
valderiana) BDU 10121, c. Oscillatoria salina (O. salina) BDU 10412,
and d. Chlorella sp.

2.2. Growth profile of algal species

Growth of selected strains was determined based on dry cell

weight (DCW) of the cultures. For DCW, known amount of algal
suspension was centrifuged at 6000 rpm for 10 min and the
pelleted algal cells were quick washed twice with water and
dried at 60 � C in hot air oven till
 Fig. 4. FTIR spectra of PHA (a) L. valderiana (b) S. elongates.

consistent weight was seen. Then, DCW was calculated from Chlorella sp., O. salina, L. valderiana and S. elongatus, the following
-1 procedure was carried
gravimetrically and expressed in g L . All the DCW measurement
was taken every three days interval during the course of algal
growth. Based on the growth of
the strains, cultures were further scaled-up in ASN III to
generate biomass for PHA extraction.

2.3. PHA extraction

Algal species were harvested at the end of stationary phase of

their growth by centrifugation as mentioned in DCW estimation.
The bio- masses were water washed to eliminate medium residual
or salts if any present along with the biomass. For PHA extraction
out. A known weight of dried biomass was added with 4 %
sodium hypochlorite solution and incubated for 30 min at 45 � C.
The sample was then centrifuged at 6000 rpm for 30 min and the
pellet was added with hot chloroform and kept overnight and
then it was precipitated with cold methanol. The precipitated
polymer was then centrifuged at 6000 rpm for 30 min to obtain
a pellet. The pellet was dissolved again in hot chloroform and
dried at 60 � C and weighed (Costa et al., 2018b).

2.4. Fourier-transform infrared spectroscopy (FTIR) analysis of PHA

samples

Qualitative analysis of PHA sample extracted from Chlorella

sp., O. salina, L. valderiana and S. elongatus was carried out by FTIR.
FTIR
 Fig. 5. TGA of PHA (a) Chlorella sp. (b) O. salina (c) L. valderiana (d) S. elongatus.

Table 2 S. elongatus was monitored in terms of DCW. The DCW of all the cultures was presented in Fig. 1 & Fig. 2.
Thermal properties of PHA samples. For S. elongatus, the maximum growth was obtained on the 18th day with a DCW of 0.32 g/L. The
PHA SAMPLES freshwater
Tg � C Tm � C
S. elongatus UTEX 2973 and Synechococcus PCC 7942 yielded 0.87 g/L and 0.33 g/L dry weight within
PHA from Chlorella sp. 6 90
PHA from O. salina 16 h 9.35
from the initial dry weight
116 of
PHA from L. valderiana 4 79 0.13 g/L and 0.12 g/L respectively under high light illumination
PHA from S. elongatus 10 94 and CO2 supply (Yu et al., 2015). For L. valderiana, maximum
growth was observed on the 18th day with DCW of 2.75 g/L,
while for O. salina the
analysis of the samples was done using Perkin Elmer Spectrum 3.1. Growth pattern of algae
(Version 10.03.09) spectrophotometer and the bands were observed in
-1
the region between 4000 and 400 cm . FTIR is used to determine The growth of cultures Chlorella sp., O. salina, L. valderiana and
the chemical
groups in PHA samples.

2.5. Thermal analysis of PHA samples

The thermal properties of polyhydroxyalkanoates synthesized

from Chlorella sp., O. salina, L. valderiana and S. elongatus were
obtained from Thermo Gravimetric Analysis (TGA) and Differential
Scanning Calo- rimetry (DSC). TGA was done in Perkin Elmer
Pyris 13 from 35 � C to 500 � C with a heating rate of 10 � C/min in
Nitrogen atmosphere and DSC was conducted — in Perkin Elmer Pyris
6 from 20 � C to 440 � C at 10 � C/ min in a nitrogen
atmosphere.

3. Results and discussion

higher growth obtained was 0.99 g/L on day 18. Reports on
character- ization of cyanobacterial strains for high biomass
production have shown the dry biomass yields for Synechococcus
PCC 7942, Oscillatoria sp., Lyngbya sp. as 1.09 g/L, 0.76 g/L, 0.64
g/L respectively after 19 days of batch cultivation (Patel et al.,
2018). In the case of tested green alga Chlorella sp., higher dry
weight was obtained on day 21 with 2.74 g/L. In a study by Thuy
et al., (2019) for production of biomass and lipid from green alga
Chlorella sp., the maximum growth was observed on 16th day with
a DCW of 1.1 g/L. Another study reported 0.15 g/L DCW for
marine
C. vulgaris after 48 h of cultivation in outdoor raceway pond
(Mathimani et al., 2017). Colla et al., (2007) studied the growth of
Spirulina platensis
under 30 � C and 35 � C in Zarrouk’s medium and the biomass
concen- tration obtained was 0.82–0.92 g/L and 0.59–0.65 g/L
respectively.
Gonçalves et al., (2019) studied the biochemical composition of
Pseu- doneochloris marina under different light and temperature
conditions and the highest biomass obtained was DCW of 1.8
g/L.

3.2. FTIR spectra of PHA samples

FTIR spectra of four PHA samples were shown in Fig. 3 & Fig.
4. The
 Fig. 6. DSC of PHA (a) Chlorella sp. (b) O. salina (c) L. valderiana (d) S. elongatus.

PHA samples were extracted from Chlorella sp., O. salina, L. represents the vibration of –CH3 group; C– O stretching at of ester-
valderiana and S. elongates were designated as PHA I, PHA II, PHA -1
carboxylic group obtained at 1637 cm , which confirmed the
III and PHA IV respectively. There is a prominent and broad signal presence of scl-PHA.
-1
at 3339 cm for PHA Characterization using FTIR was performed on the PHA obtained
-1 -1
I and III, 3348 cm for PHA II and 3349 cm for PHA IV which from
repre- Synechocystis salina (Meixner et al., 2018). The spectra obtained showed
sents the stretching of –OH within the carboxyl groups. The
-1 -1
presence of peak around 2900 cm ie., 2921 cm for PHA I and
-1 -1
II, 2923 cm for PHA III and 2919 for cm for PHA IV represents
C–H stretching vibra- tion of methyl and the methylene groups in
the polymer. The stretching of C– O group was seen from the signals
-1 -1 -1
1625 cm for PHA I, 1644 cm for PHA II and 1639 cm for PHA III
-1
and IV. Absorbance in the region of 1400 cm represents the
asymmetric and symmetric stretching of methyl group present
-1 -1
in the PHA samples. The peaks at 1025 cm , 1015 cm and
-1 –
1023 cm indicates the C O stretching in ester group.
Characteristic signals observed above confirm the presence of PHA
in all the algal samples. Further, data of this present study is further
supported by the various research works carried out by other
researchers mentioned below. The FTIR studies conducted for
the PHA sample
synthesized from Halogeometricum borinquense strain E3 by Salgaonkar
-1
and Bragança (2015) showed peak at 3585 cm which represents the
-1
O–H stretching and a peak around 2900 cm corresponds to C–
H stretching vibration. Extraction of PHA from the microalgae
Botryo-
coccus braunii was conducted (Kavitha et al., 2016b). FTIR analysis
and transmittance bands were identified at: 2933 cm—1, which
 -1 -1 -1 -1
signals 2975 cm and 2932 cm which represents the at 3278 cm represented the hydroxyl group, signals at 2920 cm
-1
-1
presence of methyl and methylene groups, 1453 cm confirmed and 2294 cm corresponds to the C–H stretching vibration of
-1
the presence of asymmetric and symmetric stretching of the methyl methyl and the methylene groups in the polymer around 1700 cm
groups and a signal indicated the CO
-1 -1
at 1721 cm confirmed the presence of carbonyl stretching of an stretching in ester bond and at 1415 cm represented the asymmetrical
ester stretching vibration in CH3 group.
group. Samantaray et al. (2011) studied the FTIR spectrum of the PHA
extracted from Aulosira fertilissima. The signal obtained around
-1 -1 3.3. Thermal analysis of PHA samples
3000 cm represents the –OH group. The peak at 1460 cm
corre- sponded to the asymmetrical stretching in the CH3 group.
-1 Thermal degradation of PHA samples extracted from Chlorella sp.,
The band at 1720 cm indicated carbonyl stretching of the ester O. salina, L. valderiana and S. elongatus was studied. Fig. 5 shows the
bond. FTIR spectra
TGA graph of PHA samples. There is an initial weight loss up to a
of the PHA extracted from Spirulina sp. LEB-18 and S. subsalsus was
temperature of � 150 � C for all the four samples due to presence
studied (Costa et al., 2018a). The band region around 1710-1750
-1 of water molecules and also presence of impurities during the PHA
cm confirmed the presence of ester carbonyl group. Kavitha et
extraction process. The thermal degradation of polymer started at
al. (2016a)
217 �C, 238 �C, 261 �C and
studied the FT-IR spectrum of Arthrospira platensis in which the peak

249 � C for PHA I, PHA II, PHA III and PHA IV respectively. The
degra- dation rate of PHA depends upon different monomer units. real time value of PHA for application studies. In future studies,
Costa et al. (2018a) studied the thermal degradation of the PHA pathway engineering or metabolic system studies of algae can be
extracted from Spirulina sp. LEB-18 and S. subsalsus and it was undertaken to overexpress the gene to accumulate PHA. In order to
found that the onset of degradation of the polymer was around put the algal bio- plastic in a sustainable path, meticulous and
250 � C and 287 � C respectively and it was concluded that the myriad R & D attempts need to be pursued in the avenues of
polymers obtained was thermally stable below a temperature of feedstock improvement, lucrative extraction, and PHA
250 � C and the melting temperature obtained from DSC was productivity.
around 171 and 173 � C for Spirulina sp. LEB-18 and
S. subsalsus respectively. The thermal degradation of PHA extracted Acknowledgement
from diazototrophic cyanobacterium Nostoc muscorum Agardh was
Authors thank National Repository for Microalgae and
identified at a temperature of 223 � C (Bhati and Mallick, 2015).
Cyanobac- teria (NRMC), Bharathidasan University, Tiruchirappalli
The Tg and Tm of the PHA polymers obtained was around 0.8 �C
for providing algal cultures.
and 178 �C. There was a decrease in both temperatures as there
was change in the polymer composition. The degradation of the
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