Bioresource Technology 265 (2018) 456–463

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Innovative polyhydroxybutyrate production by Chlorella fusca grown with T

pentoses
⁎
A.P.A. Cassuriagaa, B.C.B. Freitasa, M.G. Moraisb, J.A.V. Costaa,
a
College of Chemistry and Food Engineering, Federal University of Rio Grande, Laboratory of Biochemical Engineering, Rio Grande, RS, Brazil
b
College of Chemistry and Food Engineering, Federal University of Rio Grande, Laboratory of Microbiology and Biochemistry, Rio Grande, RS, Brazil

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

Keywords: The current study aimed to evaluate if the addition of pentoses along with variations in light intensity and
Biopolymer photoperiod can stimulate the production of polyhydroxybutyrate (PHB) and other biomolecules by Chlorella
Chlorella fusca LEB 111. The variables evaluated were the addition of xylose and arabinose as sources of organic carbon,
Luminosity different photoperiods (18 h, 12 h and 6 h light) and variations in light intensities (58, 28 and
Photoperiod
9 μmolphotons m−2 s−1). The highest PHB accumulation (17.4% w w−1) and protein production (53.2% ww−1)
Xylose
were observed in assays with xylose addition and a photoperiod of 6 h of light provided at 28 and
58 μmolphotons m−2 s−1, respectively. The highest lipid content (24.7% w w−1) was obtained with 18 h of light.
The current study contributes to the development of sustainable alternatives for the use of wastes and the
production of biomolecules from algae.

1. Introduction
Non-biodegradable plastics accumulate in the environment at a rate
of 25 × 106 ton/per year, which poses great risks to marine ecosystems
(Lohr et al., 2017). It is estimated that plastic wastes of petrochemical
origin are responsible for the death of almost 1 million animals per
year, from birds to mammals, all over the planet (Kavitha et al., 2016).
Thus, there is a demand for biodegradable polymers, which has led to
the search for species or varieties of microorganisms that are capable of
synthesizing high amounts of these compounds (Morais et al., 2017).
The polyhydroxyalkanoates (PHAs) produced from microbial
sources are materials that are compatible with the environment, as well
as being useful in medical and food applications due to their similar
properties to polymers of petrochemical origin (Martins et al., 2014).
PHAs can be classified according to the lengths of their side chain does,
polyhydroxybutyrate (PHB) is an apolar biopolymer and poorly
permeable to O2, H2O, and CO2, optically active and chiral center
having the R groups located on the same side of the polymer chain
(Silva et al., 2018). Biopolymers of microalgal origin, such as PHB and
its copolymers, have similar mechanical properties when compared to
those of petrochemical origin. The mechanical, physical and chemical
properties are similar to polymers such as polyethylene and poly-
propylene these being non-degradable polymers (Tokiwa et al., 2009).
The total degradation of materials biopolymeric (PHB) by the action of
microorganisms results in the release of CO2 and H2O by aerobic route
and CH4 by anaerobic route. This fact strengthens the possibility of
complementation and/or substitution of the polymer of petrochemical
origin (Morais et al., 2017). Studies are widely reported for PHA pro-
duction with strains of Pseudomonas pseudoflavas, Pseudomonas paller-
onii (Reddy et al., 2017), γ-Proteobacteria, Cellvibrio sp., and Pseudo-
monas sp. (Huang et al., 2016). The production of PHA's is also
described for microalgae where approximately 100 strains of micro-
algae and cyanobacteria have been examined, with PHB concentrations
varying from 0.04% to 6% (w w−1) in their dry biomass (Kavitha et al.,
2016). The pathways reported for PHB production have been de-
termined for some cyanobacteria such as Spirulina sp. (Martins et al.,
2014), Synechococcus (Miyake et al., 2000) and Synechocystis (Khetkirn
et al., 2016). Studies about the production and identification of PHB in
Chlorophyta are still recent. Kavitha et al. (2016) identified low con-
centrations of this polymer in Chlorella vulgaris, Botryococcus braunii and
Chlorococcum humicola.
Microalgae of the Chlorella genus have been used since the 1960s for
large scale cultivation for food supplementation. In addition, the re-
presentatives of this genus have shown potential to produce biomass for
biofuels production (Rasoul-Amini et al., 2014).
One of the great challenges involved in the production of PHB by
microalgae is the carbon source since about 50% of the dry biomass is
related to this source. However microalgal cultures have high

⁎
Corresponding author at: Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, P.O. Box 474, 96203-900, Av. Itália,
km 8, Rio Grande, RS, Brazil.
E-mail address: jorgealbertovc@terra.com.br (J.A.V. Costa).

https://doi.org/10.1016/j.biortech.2018.06.026
Received 15 May 2018; Received in revised form 8 June 2018; Accepted 10 June 2018
Available online 11 June 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
A.P.A. Cassuriaga et al.
versatility as to the absorption of nutrients supplied. In addition, mi-
croalgae can use solar energy, CO2, and H2O to synthesize organic
molecules through photosynthesis (Slocombe and Benemann, 2016). In
view of this, the use of organic and inorganic carbon sources such as
pentoses and CO2 may be alternatives for reducing biomass production
costs (Freitas et al., 2017b). From this, it is possible to indicate the
possibility of production of PHB with CO2 this is likely to contribute to
reducing the environmental effects caused by CO2 emissions (Slocombe
and Benemann, 2016). Several studies have shown that different species
of Chlorella can consume various carbon sources among them the
pentoses. Moreover, polymer production studies by these microalgae is
limited (Kavitha et al., 2016). The large availability of lignocellulosic
wastes containing high amounts of xylose and arabinose generates a
demand for different biological agents that are capable of utilizing these
sugars. Studies using pentoses (D-xylose and L-arabinose) have been
reported; for example, Zheng et al. (2014) reported metabolic absorp-
tion routes with Chlorella sorokiniana cells. Leite et al. (2015) verified
that the addition of D-xylose in cultures of Chlorella sp. favored the
increase of lipid storage. Freitas et al. (2016) reported that the use of
20 mg L−1 of xylose triggered an increase in the efficiency of photo-
system II (PSII) in C. minutissima, in addition to the increase in bio-
molecules such as carbohydrates (Freitas et al., 2017a,b), with potential
for bioethanol production.
Microalgae use light as a source of energy during photosynthesis to
produce various compounds. The mechanisms involved in photo-
synthesis, such as the activation or inactivation of photosystems, can be
modulated by modifications in light periodicity and intensity varia-
tions. These modifications are considered efficient strategies to stimu-
late microalgal growth and the accumulation of specific biomolecules
(Krzeminska et al., 2014). Furthermore, these modifications cause a
synergistic effect that can stimulate the accumulation of reserve com-
pounds, such as lipids, which are precursors for biopolymer production
(Leite et al., 2016).
Therefore, the use of microalgae for specific biomolecule production
has attracted much attention due to high rates of microalgal growth and
the capacity to use different substrates (Freitas et al., 2016). To provide
data to help in the understanding about the production of PHB and
biomolecules as protein, carbohydrate and lipids by microalgae, the
present study evaluated the effects of pentose addition in association
with variation of light cycles and light intensity in cultures of Chlorella
fusca.
2. Material and methods
2.1. Microalgae and culture conditions
Chlorella fusca LEB 111 (Duarte et al., 2016), from the Collection of
the Laboratory of Biochemical Engineering of the Federal University of
Rio Grande (FURG), Rio Grande do Sul, Brazil, was used.
The microalgae were cultured in BG-11 medium (Rippka et al.,
1979) composed of (g L−1) Sodium nitrate (NaNO3) (1.5), Potassium
phosphate monoacid heptahydrate (K2HPO4·7H2O) (0.04), Magnesium
sulfate heptahydrate (MgSO4·7H2O) (0.075), Calcium chloride dehy-
drate (CaCl2·2H2O) (0.036), Ferric ammonium citrate (C6H11FeNO7)
(0.006), Sodium carbonate (Na2CO3) (0.02), Nitric acid (C6H8O7)
(0.006) and 1 mL of A5 + Co solution (g L−1): Boric acid (H3BO3)
(2.86), Manganese(III) chloride tetrahydrate (MnCl2·4H2O) (1.81), Zinc
sulfate heptahydrate (ZnSO4·7H2O) (0.222), Sodium molybdate
(NaMoO4) (0.015), Copper(II) sulfate pentahydrate (CuSO4·5H2O)
(0.079) and Cobalt (II) nitrate hexahydrate (Co(NO3)·6H2O) (0.0494).
Cultures were exposed to different light intensities and photo-
periods, a 50% reduction in the nitrogen source and replacement of the
carbon source (Na2CO3 0.02 g L−1) by D-xylose and L-arabinose (Vetec
Química, Sigma-Aldrich Corporation). The assays were conducted in
duplicate in Erlenmeyer photobioreactors with a working volume of
1.8 L for 10 days.
457
Bioresource Technology 265 (2018) 456–463
2.2. Addition of pentoses, variation of light intensity and photoperiod
The assays with Chlorella fusca LEB 111 were exposed to 40 W
fluorescent light bulbs in a thermostatted greenhouse set to 30 °C. The
cultivations were exposed to light intensity variations (58, 28 and
9 μmolphotons m−2 s−1), light periodicity (18 h: 6 h, 12 h: 12 h, 6 h: 18 h
in cycles light: dark) and the addition of D-xylose and L-arabinose (Vetec
Quimica, Sigma, Aldrich Corporation). For cultures with pentose ad-
ditions, the nitrogenous compound (NaNO3) was reduced by 50%
(0.75 g L−1) along with the removal of the standard carbon source
(Na2CO3) from the BG-11 medium. The addition of xylose and arabi-
nose was carried out by a synthetic broth containing 20 mg L−1 of
pentoses, as previously described by Freitas et al. (2016, 2017a). Con-
trol assays were performed without modification of the BG-11 medium
and without addition of pentoses. The conditions utilized in this study
are listed in Table 1.
2.3. Biomass concentration
The biomass concentration was monitored daily by measuring the
optical density of the cultures with a spectrophotometer (QUIMIS
Q798DRM, Diadema – SP – Brazil) at 670 nm, using a previously de-
termined standard curve of Chlorella fusca LEB 111, relating the dry
weight and the corresponding optical density (Costa et al., 2002).
2.4. Biomass volumetric productivity
The maximum biomass productivity (Pmax, g L−1 d−1) was de-
termined according to equation Pmax = (Xt − X0)/t − t0, where Xt is the
biomass concentration (g L−1) at time t (d) and X0 is the biomass
concentration (g L−1) at time t0 (d) (Bailey and Ollis, 1986).
2.5. Maximum specific growth rate
The maximum specific growth rate (μmax, d−1) was determined
using an exponential regression applied to the logarithmic growth
phase.
2.6. PHB carbon conversion factor
The substrate conversion factor (carbon source) in PHB (YS/PHB,
g g−1) was determined by using to equation YS/PHB = (P − P0)/S − S0,
where P0 and S0 representing the product concentration and the sub-
stratum concentration, respectively, at the beginning of the culture. P is
the maximum PHB concentration, and S is the final substratum con-
centration.
2.7. Determination of pH
The pH was determined by direct measurement using a portable
digital pH meter (Mettler Toledo FiveGoTM, Switzerland) (APHA,
1998).
2.8. Recovery and characterization of biomass
At the end of the cultivations, biomass was recovered by cen-
trifugation (HITACHI, himac CR-GIII, Japan) at 9,690g for 20 min, re-
suspended in distilled water and centrifuged again for 10 min under the
same conditions for improved nutrient removal. Centrifuged biomass
was frozen at −80 °C (Eppendorf, New Brunswick Scientific
InnovaU535, Brazil) for 48 h and then lyophilized (LAB CONCO, USA).
Lyophilized samples were stored at −20 °C for further characterization.
2.8.1. Characterization and quantification of polyhydroxybutyrate
Biopolymer characterization was conducted on samples taken on
the 10th day of cultivation and then subjected to methanolysis.
 A.P.A. Cassuriaga et al.

Table 1
Maximum cell concentration (Xmax, g L−1), maximum productivity (Pmax, g L−1 d−1), maximum specific growth rate (µmax, d−1), PHB production (%, w w−1), PHB carbon conversion factor (YS/PHB g g−1) and
carbohydrate (%, w w−1), protein (%, w w−1) and lipids (%, w w−1) concentrations (mean ± standard derivation) for Chlorella fusca LEB 111 cultivated in different photoperiods, light intensities (I, μmolphotons m−2 s−1)
and carbon sources.
Photoperiod 18 h light

Carbon source I Xmax Pmax µmax PHB (%) (w w−1) Y(S*/PHB) Carbohydrates (%) (w w−1) Protein (%) (w w−1) Lipids (%) (w w−1)

Control 58 0.80 ± 0.08i,j 0.09 ≤ 0,01 g 0.11 ≤ 0.01ª,b,c,d 0.5 ± 0.1ª 0.21 ≤ 0.01ab 22.6 ± 1.1 g,h 34.0 ± 0.3b,c,d,e,f,g 20.5 ± 2.9c,d,e,f,g
Xylose 58 0.80 ± 0.06j 0.06 ≤ 0.01e,f 0.12 ≤ 0.01b,c,d 3.4 ± 0.3ª 1.23 ± 0.21de 26.5 ± 0.6i 47.9 ± 0.2m 22.3 ± 0.2e,f,g,h
Arabinose 58 0.75 ≤ 0.01 h,i,j 0.05 ≤ 0.01d,e,f 0.13 ≤ 0,01c,d 1.6 ± 0.5ª 0.47 ± 0,42abc 21.2 ± 0.3f,g,h 27.1 ± 0.6ª 17.2 ± 0.2a,b,c,d
Control 28 0.48 ± 0.03d,e,f,g 0.03 ≤ 0.01b,c,d,e 0.15 ± 0,01ª,b,c,d 0.7 ± 0.3ª 0.16 ≤ 0.01a 20.3 ≤ 0.01e,f,g 29.7 ± 0.4a,b 25.5 ± 0.7 h
Xylose 28 0.52 ≤ 0.01d,e,f,g 0.03 ≤ 0.01ª,b,c 0.08 ± 0,01ª,b,c 3.5 ± 1.0a 0.91 ± 0.34 cd 24.5 ≤ 0.01 h,i 33.3 ± 1.0b,c,d,e,f 18.8 ± 1.1b,c,d,e,f
Arabinose 28 0.60 ≤ 0.01f,g,h,i 0.03 ≤ 0.01ª,b,c 0.11 ± 0,01ª,b,c,d 14.0 ± 1.4c,d 4.15 ± 0.50i 19.2 ± 0.7d,e,f,g 29.4 ± 0.7a,b 16.0 ± 0.2a,b,c
Control 9 – – – 0.5 ± 0.1ª – 20.6 ± 1.6e,f,g 31.5 ± 0.2a,b,c,d 16.4 ± 0.6a,b,c,d
Xylose 9 0.44 ≤ 0.01b,c,d,e,f,g 0.02 ≤ 0.01ª,b 0.13 ± 0,03b,c,d 16.2 ± 0.8d 3.45 ≤ 0.01 h 18.6 ± 0.1c,d,e,f 29.5 ± 0.7a,b 24.7 ± 0.2 g,h
Arabinose 9 0.35 ≤ 0.01a,b,c,d,e 0.04 ≤ 0.01ª,b 0.20 ± 0,03f 4.9 ± 0.5ª 0.85 ≤ 0.01bcd 17.0 ± 0.1c,d,e 33.9 ± 0.2b,c,d,e,f,g 17.9 ± 0.3a,b,c,d,e

Photoperiod 12 h light

Control 58 0.84 ± 0.02j 0.06 ≤ 0.01f,g 0.13 ≤ 0.01c,d,e 5.5 ± 0.2ª 2.05 ± 0.07f 21.8 ± 0.3f,g,h 42.6 ± 0.9j,k,l 19.4 ± 0.4b,c,d,e,f
Xylose 58 0.53 ± 0.06d,e,f,g,h 0.05 ≤ 0.01d,e,f 0.16 ≤ 0.01d,e,f 5.5 ± 1.4ª,b 1.15 ± 0.82de 19.5 ± 0.4d,e,f,g 35.5 ± 0.9c,d,e,f,g,h 19.5 ≤ 0.01b,c,d,e,f

458
Arabinose 58 0.57 ± 0.02f,g,h 0.05 ≤ 0.01c,d,e,f 0.10 ≤ 0.01ª,b,c,d 10.7 ± 1.1c 3.05 ± 0.58gh 21.9 ± 0.1f,g,h 38.5 ± 0.2 g,h,i,j 19.4 ± 0.4b,c,d,e,f
Control 28 0.57 ± 0.05f,g,h 0.06 ± 0.03f 0.10 ≤ 0.01ª,b,c,d 2.1 ± 0.3ª 0.58 ± 0.05abc 11.5 ≤ 0.01ª 41.9 ± 0.3i,j,k,l 16.2 ± 0.9a,b,c,d
Xylose 28 0.63 ± 0.02 g,h,i,j 0.06 ≤ 0.01d,e,f 0.23 ± 0.01e,f 2.7 ± 0.5ª 0.83 ± 0.07bcd 20.6 ± 0.1e,f,g 37.1 ± 1.2e,f,g,h,i 19.2 ≤ 0.01b,c,d,e,f
Arabinose 28 0.59 ≤ 0.01c,d,e,f,g 0.05 ≤ 0.01e,f 0.11 ≤ 0.01ª,b,c,d 1.6 ± 0.1ª 0.46 ± 0.03abc 16.4 ≤ 0.01b,c,d 37.6 ± 1.8f,g,h,i 18.4 ≤ 0.01b,c,d,e,f
Control 9 – – – 2.9 ± 0.8ª – 17.0 ± 0.9c,d,e 40.0 ≤ 0.01 h,i,j,k 16.0 ± 0.3a,b,c
Xylose 9 0.39 ≤ 0.01a,b,c,d,e,f 0.01 ± 0.02ª,b,c 0.06 ± 0.01ª,b 10.2 ± 1.5b,c 1.97 ± 0.27ef 15.0 ± 1.4a,b,c 32.8 ≤ 0.01b,c,d,e 22.1 ± 2.8e,f,g,h
Arabinose 9 0.33 ± 0.01a,b,c,d 0.02 ± 0.01ª,b 0.05 ± 0.01ª 4.6 ± 0.4ª 0.76 ± 0.03abcd 19.4 ± 0.3d,e,f,g 38.0 ± 0.3f,g,h,i,j 18.2 ± 0.9a,b,c,d

Photoperiod 6 h light

Control 58 0.60 ≤ 0.01e,f,g,h 0.04 ≤ 0.01b,c,d,e 0.10 ≤ 0.01ª,b,c,d 5.3 ± 1.0a,b 0.78 ± 0.17abcd 18.6 ± 0.1c,d,e,f 46.4 ± 1.9 l,m 22.9 ≤ 0.01f,g,h
Xylose 58 0.30 ≤ 0.01a,b,c 0.01 ≤ 0.01ª,b 0.10 ≤ 0.01ª,b,c,d 5.3 ± 0.4ª,b 0.55 ± 0.05abc 13.1 ± 0.3a,b 53.2 ± 0.3n 18.3 ± 0.9b,c,d,e
Arabinose 58 0.18 ≤ 0.01ª 0.01 ≤ 0.01ª – 5.3 ± 1.1ª,b 0.76 ± 0.16abcd 16.8 ± 0.6b,c,d,e 35.1 ± 1.3c,d,e,f,g 14.9 ± 0.6a,b
Control 28 0.29 ± 0.01a,b,c 0.04 ≤ 0.01ª,b 0.07 ± 0.01ª,b,c 1.6 ± 0.6ª 0.32 ≤ 0.01abc 38.7 ± 0.4j 40.9 ± 0.3i,j,k 17.0 ± 0.3a,b,c,d
Xylose 28 0.21 ≤ 0.01ª 0.03 ≤ 0.01ª – 17.4 ± 1.5d 2.56 ± 0.31 fg 16.2 ± 0.4b,c,d 31.0 ± 1.3a,b,c 20.7 ± 0.9d,e,f,g
Arabinose 28 0.28 ≤ 0.01a,b 0.06 ≤ 0.01d,e,f 0.12 ± 0.01c,d 3.1 ± 0.6ª 0.25 ± 0.03ab 21.9 ± 0.1f,g,h 32.1 ≤ 0.01b,c,d 18.6 ± 0.8b,c,d,e,f
Control 9 0.28 ≤ 0.01a,b 0.02 ≤ 0.01ª,b,c – 5.7 ± 0.8ª 0.52 ± 0.48abc 21.6 ± 1.7f,g,h 43.6 ± 0.5 k,l,m 15.3 ± 0.7a,b
Xylose 9 0.26 ± 0.02ª 0.01 ≤ 0.01ª,b 0.10 ≤ 0.01ª,b,c,d 5.3 ≤ 0.01ª,b 0.57 ± 0.02abc 19.3 ≤ 0.01d,e,f,g 36.0 ± 0.4d,e,f,g,h 15.0 ± 0.8a,b
Arabinose 9 0.23 ≤ 0.01ª 0.01 ≤ 0.01ª,b 0.07 ≤ 0.01ª,b,c 1.9 ± 0.3ª 0.24 ± 0.09ab 21.4 ± 2.0f,g,h 45.9 ± 2.9 l,m 13.5 ± 0.1a

Control: 0.02 g L−1 Na2CO3; Xylose: 20 mg L−1 D-xylose; Arabinose: 20 mg L−1 L-arabinose. *S: Carbon source (Na2CO3 or D-xylose or L-arabinose). **Means followed by the same superscript letter in the same column
indicates no significant difference at a 99% confidence level (p ≤ 0,01). ***Lines with the – symbol represent cell death, without quantification being possible.
Bioresource Technology 265 (2018) 456–463
A.P.A. Cassuriaga et al.
Methanolysis was performed using 15 mg of biomass that had pre-
viously undergone pigments extraction with 99.8% (v v−1) methanol,
according to the method proposed by Lichtenthaler (1987). After pig-
ment removal, the biomass was dried (105 °C/24 h), and methanolysis
was carried out with 1 mL of chloroform, 0.85 mL of methanol, and
0.15 mL of sulfuric acid at 100 °C for 3.5 h (Brandl et al., 1988). After
methanolysis, the methyl ester groups formed were analyzed in a gas
chromatograph (2014 Shimadzu, Japan), equipped with a Restek-Rtx
silica capillary column (30 m, 0.25 mm, 0.25 μm) and flame ionization
detector (FID). Hydrogen was used as carrier gas with an injection
volume adjusted to 1 μL, a column temperature gradient of 60 °C for
2 min with a ramp rate from 25 °C/min to 180 °C/8 min. The polymer
concentration was calculated using a standard curve constructed using
a solution of poly (3-hydroxybutyric-co-3-hydroxyvaleric acid) con-
sisting of 88 mol% HB and 12 mol% HV (Sigma Aldrich) (Zhang et al.,
2015).
2.8.2. Protein and carbohydrate concentration
Protein and carbohydrate analyses were performed on biomass ex-
tracts (5 mg/10 mL distilled water) that had been ultrasonicated (COLE
PARMER CPX 130 – Illinois – USA). The total carbohydrate con-
centration in biomass was determined using the phenol–sulfuric
method, with a standard glucose curve (Dubois et al., 1956). Protein
content in biomass was determined using the colorimetric method
proposed by Lowry et al. (1951), with a standard curve of bovine serum
albumin. The lipid concentration in the biomass was determined using
chloroform and methanol as solvents (Folch et al., 1957).
2.8.3. Protein profile
Proteins were extracted every 48 h by the addition of sample buffer
(4× sample buffer: 80 mmol L−1 Tris/HCl, pH 6.8; 2% (w v−1) sodium
dodecyl sulfate [SDS]; 15% (v v−1) glycerol; 0.006% (w v−1) purple m-
cresol; 0.1 mol L−1 2-mercaptoethanol) and heated for 5 min at 100 °C,
followed by centrifugation at 10,000g for 1 min. The samples were then
subjected to discontinuous SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) according to Laemmli (1970), using a 5% acrylamide
stacking gel and a 12.5% acrylamide resolving gel.
2.9. Statistical analyses
The differences between the average values obtained from each
assay were assessed through analysis of variance, followed by Tukey’s
test with a confidence level of 99% (p > 0.01).
3. Results and discussion
3.1. Microalgal growth kinetics
C. fusca, when submitted to variations of light intensity in the same
photoperiod (12 h of light) presented different growth profiles (Fig. 1).
Leite et al. (2015) evaluated the addition of pentoses (xylose) in
Chlorella cultures and observed that the strain was able to reach a
maximum growth of 0.33 g L−1 and 0.19 g L−1 in mixotrophic and
heterotrophic cultures, respectively.
Freitas et al. (2016), where the highest cell densities were obtained
with 50% of the recommended level of nitrogen compound (0.75 g L−1
NaNO3) and the addition of pentoses (D-xylose and L-arabinose). The
same effect of the C/N ratio occurs in C. fusca cultures, once the ad-
dition of xylose and 18 h of light improved the cell growth (0.80 g L−1,
this being the maximum growth value), followed by the assays with
arabinose (0.75 g L−1) using the same photoperiod, all subjected to
nitrogen reduction (NaNO3) (Table 1).
Xu et al. (2016) attributed light intensity as the factor that may be
responsible for modifying cell growth in mixotrophic cultures, due to
the metabolites involved in cell turnover rates. Changes in light periods
produce biochemical alterations, such as the activation of photosystems
459
Bioresource Technology 265 (2018) 456–463
for longer periods, resulting in higher growth rates, as we observed in
present study under all conditions and as reported by Khoeyi et al.
(2012). These authors, using a light period of 16 h and light intensities
of 37.5 and 62.5 μmolphotons m−2 s−1, obtained biomass concentration
increases by C. vulgaris (0.6 g L−1 and 2.0 g L−1, respectively).
Our results suggest that the BG-11 medium without pentose sup-
plementation was not able to supply the nutritional demand for organic
carbon at low luminous intensities during the growth of Chlorella fusca
(Fig. 1). This behavior was confirmed by the maximum biomass con-
centration for Chlorella fusca (0.63 g L−1) in the photoperiod of 12 h
light with xylose addition and a light intensity 28 μmolphotons m−2 s−1
(Table 1). According to Zheng et al. (2014), these results are typical of
mixotrophic cultures that undergo different growth responses because
this form of cultivation triggers modifications in the mechanisms of
absorption of the carbon sources. The nitrogen in the medium is re-
quired for several substances of primary microalgal metabolism
(Rosenberg et al., 2008) and by reducing the nitrogen compound by
50% (NaNO3 0.75 g L−1) results in conditions that do not seem to
supply the necessary demand for the cellular growth of Chlorella fusca
when grown under the lowest luminous intensities.
The biomass yield in photoperiod 18 h of light and
58 μmolphotons m−2 s−1 was 0.06 g L−1 d−1 for xylose, being this value
higher than the control conditions of Krzeminska et al. (2014), with
Chlorophyta of the genus Botriococcus braunni (0.034 g L−1 d−1) when
light intensity variation similar to that of the present study was pro-
vided. The highest specific growth rate was achieved with the light
intensity condition of 28 μmolphotons m−2 s−1 and photoperiod 12 h of
light along with the addition of xylose (0.23 d−1) and is statistically
different (p ≤ 0.05) from the others, with the exception of the experi-
ments with arabinose (0.20 d−1), 18 h of light and less light intensity
and for those with xylose (0.16 d−1),with 12 h of light and
58 μmolphotons m−2 s−1.
In the assays the pH increased did not increase significantly during
the 10 days of the experiment, being between 9.5 and 10.1 in all studied
conditions (control and pentoses). Rosa et al. (2018) in culture with
MEA (monoethanolamine) and the same strain of the present study,
obtained values around 9.2–9.7. In both studies, the high pH values
observed were beneficial for the cultivation of C. fusca LEB 111, since
this microorganism was isolated from the ash stabilization pond of a
thermoelectric plant, this place with pH above 9.0.
The growth results (Table 1) show the positive effect in the non-
occurrence of cell death at the lowest luminous intensities studied (but
these do not differ statistically from the other conditions).
3.2. PHB production
The effects of the modification of culture parameters on Chlorella
strains are still rarely reported (Kavitha et al., 2016). We expect this
study to be a precursor in identifying possible new ways and modes to
optimize PHB production. According to Morais et al. (2017) and
Martins et al. (2014), to increase PHB production, a high level of lipid
accumulation in microalgal biomass is required.
According to Markou and Nerantzis (2013), variations in the bio-
polymer concentration are directly affected by the cellular growth of
the microorganism. In general, the lower at cell growth of a micro-
organism, a greater at the tendency of polymer accumulation in the
biomass. This fact can be attributed to the lower consumption of the
carbon source by the microalgae for its growth. The highest PHB con-
centration observed in the present study (17.4% w w−1) was obtained
in response to microalgae metabolism during a lower level of cell
growth (0.21 g L−1 – photoperiod of 6 h of light). Kavitha et al. (2016)
obtained similar results as in the present study, i.e., low cell growth
yielded 16.4% PHB (also quantified via chromatography) in Bo-
tryococcus braunii grown in 60% of sewage as the growth medium.
When supplied with a photoperiod of 12 h light,
58 μmolphotons m−2 s−1 of light intensity and arabinose, the microalga
A.P.A. Cassuriaga et al.
Chlorella fusca LEB 111 presented a PHB concentration of 10.7%
(w w−1) (Table 1). The reduction of the exposure times to light resulted
in PHB accumulation, indicating the potential for a reduction in costs
related to microalgal cultivation.
According to Zheng et al. (2014), for the assimilation of pentoses by
Chlorophytes to occur, the D-xylose transport mechanisms are necessary
as well as the high-affinity systems in conjunction with D-glucose
transporters for non-native species. These mechanisms release NADH
molecules, leaving this reduced molecule available for possible meta-
bolism and synthesis of PHB. For PHB synthesis to occur, the conversion
of acetoacetyl-CoA by the acetoacetyl-CoA reductase enzyme requires a
gradient of reduced molecules for D-3-hydroxybutyrate, the precursor
compound of the polyhydroxybutyrate molecule (Sudesh et al., 2000;
Baroukh et al., 2016). Thus, the release of NADH molecules resulting
from the metabolism of pentoses may lead to the stimulation of polymer
production, which is still little explored in Chlorophytes, where these
described metabolic pathways predict reactions that may be occurring
intracellularly.
The metabolism of photosynthetic microorganisms that are sub-
jected to photoperiod variation together with a reduction in the ni-
trogen supply can converge to the accumulation of reserve lipids, such
as PHB (Morais et al., 2017). When the Chlorella fusca cultures were
subjected to photoperiod modification with cycles of 18 h of light and
light intensity 9 μmolphotons m−2 s−1, the strain showed a capacity to
accumulate PHB in higher concentrations of 16.2% (w w−1) with xylose
addition (Table 1). In this study, besides pentose additions, the nitrogen
reduction in Chlorophyta cells possibly triggered the conversion of
microalgal metabolism to reduce protein production, which resulted in
the accumulation of ATP. According to Hondo et al. (2015) and Wu
et al. (2006), when ATP is in excess, this can lead to oxidative phos-
phorylation, leading to the accumulation of precursor co-enzymes of
PHB production.
It is believed that the phases of photosynthesis are directed ac-
cording to the light supply, i.e., the photochemical phase is light-de-
pendent, and the dark phase (or the biochemical phase) is light
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Bioresource Technology 265 (2018) 456–463
Fig. 1. Growth profile of Chlorella fusca LEB 111 for
the assays: Control (Na2NO3) (■), Xylose (▴) and
Arabinose (●), 50% nitrogen in a photoperiod of
18 h light and light intensity of (a)
58 μmolphotons m−2 s−1 (b) 28 μmolphotons m−2 s−1
and (c) 9 μmolphotons m−2 s−1; photoperiod of 12 h
light with light intensity of (d)
58 μmolphotons m−2 s−1 (e) 28 μmolphotons m−2
s−1 e (f) 9 μmolphotons m−2 s−1.
independent. Compounds that are produced in the light-dependent
phase are used in the dark phase for synthesis of essential molecules
used for growth even under stress conditions (Bouterfas et al., 2006).
This fact was observed for the assays with xylose and arabinose addi-
tion in the three light intensities studied, where even under low light
conditions, PHB production occurs.
Zheng et al. (2014) described the process of pentose absorption in
Chlorella sorokiniana, where non specific transporters are responsible
for D-xylose and L-arabinose trans-membrane absorption. Thus demon-
strating that the mixotrophic conditions can be maintained when using
pentoses as a carbon source for microalgal growth. In this sense, the
conversion factor of pentoses to PHB reached a higher value
(4.15 g g−1) when supplied 18 h light and light intensity
28 μmolphotons m−2 s−1, and is statistically different (p ≤ 0.05) from the
other.
The results show that PHB production by Chlorophyta should be
explored further. The metabolic pathways for obtaining this polymer
have only been consolidated for the cyanobacteria. The physical mod-
ifications of light periodicity and luminous intensity, together with the
addition of pentoses and reduction of nitrogen, indicate that the pro-
cesses developed in the present study demonstrate that the production
of PHB by Chlorella is possible.
3.3. Biomass composition
According to Table 1, the carbohydrate content (38.7% w w−1) in-
creased for the control assays under intermediate light intensity
(28 μmolphotons m−2 s−1) and 6 h of light. Supply light for 18 h at light
intensity of 58 μmolphotons m−2 s−1 promoted carbohydrate production,
resulting in level of 26.5% (w w−1), for the assay with xylose (Table 1).
Deamici et al. (2016) reported a level of 25.2% of carbohydrates in
Chlorella biomass, when this strain was grown with BG-11 medium in a
tubular photobioreactor under conditions similar to the control condi-
tions of this study. Their results are similar to the results obtained for
the control cultures (21.8% w w−1), with a light intensity of
A.P.A. Cassuriaga et al.
Fig. 2. SDS-PAGE analysis of Chlorella fusca LEB 111 grown in a photoperiod of 12 h light and a light intensity of 58 μmolphotons m−2 s−1 (a) Control, (b) Xylose and
(c) Arabinose; light intensity of 9 μmolphotons m−2 s−1 (d) Control, (e) Xylose and (f) Arabinose. *PS – Protein standards.
58 μmolphotons m−2 s−1 and a 12h light period (Table 1). These re-
sponses indicate that the Calvin cycle, which is responsible for produ-
cing sugars that will accumulate in the plastids, has been diverted to the
production of compounds other than the reserve carbohydrates or wall
components (Trivedi et al, 2015). Carbohydrate synthesis can be the
result of the stimulation of the systems involved in the Calvin-Benson
reactions, since the products of the light reactions of this cycle are ATP
and NADPH, which are used to fix carbon in sugars (Zhang et al., 2015).
The highest protein content (53.2% w w−1) was obtained for assays
with xylose, a photoperiod of 6 h and light intensity of
58 μmolphotons m−2 s−1, which was significantly higher (p ≤ 0.05)
compared to the other conditions evaluated (Table 1). The production
of higher levels of proteins is stimulated by metabolic pathways that
reduce the carbon fixation to produce carbohydrates (Maeda and
Thompson, 1986).
The oscillation in the biochemical composition of the Chlorella
biomass can be the result of changes in the photoperiods. When cultures
are exposed to shorter periods of light, this can result in changes in
membrane permeability, which influence nutrient assimilation (Maeda
and Thompson, 1986) that may stimulate biomolecule production.
The reactor type, the cultivation mode, together with factors such as
luminous intensity and photoperiod, are essential for determining
which absorption mechanism the microorganism will adopt to produce
compounds of interest (Xu et al., 2016). As demonstrated in the results
obtained for C. fusca LEB 11 and by Freitas et al. (2017a), who used a
similar luminous intensity (33.7 μmolphotons m−2 s−1) on C. minutissima
and a raceway type bioreactor, a lower protein yield (14.8% w w−1) is
produced for assays with pentoses (Table 1).
The lipid content in all treatments evaluated in the present study
were higher than those reported by Deamici et al. (2016) (13% w w−1)
for a C. fusca strain grown in BG-11 culture medium. For the assays with
xylose addition, 18 h of light supplied at a luminous intensity of
9 μmolphotons m−2 s−1 resulted in the highest lipid concentration of
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Bioresource Technology 265 (2018) 456–463
24.7% (w w−1) (Table 1). This increase in lipid content may be a
consequence of the different conditions imposed on C. fusca, such as
nitrogen and luminosity reduction and pentose addition. Studies by Xu
et al. (2016) verified that the exposure of certain crops to stress con-
ditions, such as the reduction of their nitrogen source and excess light,
may favor the increase in their lipid content, especially the triacylgly-
cerol fractions.
When microalgal cultures are exposed to stress conditions, lipid
accumulation may occur, while in optimum conditions microalgae tend
to utilize the energy available for cell multiplication (Shivani et al.,
2015). The tendency to increase the lipid contents of microalgal cells,
together with the lower production of biomass, was observed in the
present study. Seoane et al. (2015) reported that the reduction of cell
concentration increases the amount of light available to crops during
cultivation and may increase lipid accumulation.
Microalgal metabolism is a system which, when provided with op-
timum conditions of cultivation, occurs the tendency to follow ex-
ponential cell growth and in turn accumulate different molecules
(Martins et al., 2014). The metabolic routes of carbohydrate and lipid
assimilation are opposite, since when minor cell growth occurs the
microalgal metabolism becomes available for the accumulation of re-
serve molecules, such as lipids or carbohydrates (Trivedi et al, 2015).
As can be observed in the present study where the largest lipid accu-
mulation (26.5% w w−1) was obtained with addition of xylose, less
luminous intensity and 18 h light supply, being one of the conditions to
which PHB concentration increased (16.2%). The accumulation of
carbohydrates was stimulated by providing greater exposure times to
light (18 h) and light intensity of 58 μmolphotons m−2 s−1, obtaining
26.5% (w w−1). Thus, it can be observed that the results obtained in the
present study evidenced that the microalgal metabolism when being
submitted to different culture conditions like light supply and carbon
sources, modified its form of accumulation in different biomolecules.
Therefore, we conclude that the availability of carbon supplied to
A.P.A. Cassuriaga et al.
the microalga Chlorella fusca, together with the light intensity and
periodicity variation, provided conditions for the accumulation of high
concentrations of biomolecules such as carbohydrates, proteins and li-
pids in the biomass.
3.4. Protein profile
Protein profiles observed by SDS-PAGE revealed possible differences
in the experiments with pentose additions, nitrogen reduction, varia-
tion of light intensity and light periodicity over the 10 days of culture
for Chlorella fusca LEB 111 (Fig. 2).
Modification of the carbon source in conjunction with the reduction
in light periodicity (from 18 h to 12 h) and a light intensity of
58 μmolphotons m−2 s−1 triggered a possible increase in Rubisco content
(56 kDa) (Fig. 2b). In contrast, the addition of arabinose as a source of
organic carbon for the cultures of C. fusca LEB 111 resulted in the
greater degradation of this protein (Fig. 2c). Freitas et al. (2016) re-
ported that the addition of a pentose in conjunction with the use of 10%
CO2 decreased the Rubisco levels in C. minutíssima. Esquível et al.
(2006) reported that this enzyme catalyzes the fixation of carbon di-
oxide to ribulose-1,5-bisphosphate in the Calvin cycle, producing the 3-
phosphoglycerate essential for the occurrence of photosynthesis.
C. fusca LEB 111 exposed to reduced light intensity and pentose
addition displayed a possible increase in photosynthetic activity, which
can be identified by the greater visibility of Rubisco bands in assays
with xylose (well 6, 8 and 10 – Fig. 2e) and arabinose (wells 4, 6, 8 and
10 – Fig. 2f). It is believed that because the enzyme Rubisco plays a
fundamental role in the photosynthetic reaction and is characterized by
stable activity under normal conditions, stress conditions can intensify
its activity for cellular maintenance, thus making its detection at higher
levels possible (Esquível et al., 1998; Freitas et al., 2017b).
The energy absorbed by the microalgae is reflected by components
of the nucleus of photosystem II; here, the D1 and D2 subunits are re-
sponsible for carrying electrons through the thylakoid membrane
(Ferreira et al., 2000; Melis et al., 2000). Our results show more pro-
nounced bands corresponding to subunits D1 (38 kDa) and D2 (39 kDa)
when L-arabinose was added (wells 8 and 10, Fig. 2c). Fig. 2 also shows
that the most marked changes in protein profiles were achieved with
the addition of xylose and arabinose along with nitrogen reduction. It is
possible that the increase in D1 and D2 proteins (38–39 kDa) indicates a
period of higher photosynthetic activity due to the reduction of the light
intensity to 9 μmolphotons m−2 s−1. Freitas et al. (2017a) also observed
higher band intensities for proteins D1 and D2 in cultures of C. minu-
tissima exposed to a light intensity 8.44 μmolphotons m−2 s−1.
The reduction of the nitrogen concentration (by 50%) and the ad-
dition of xylose and arabinose (20 mg L−1) had a positive effect on the
protein profile, stimulating the production of enzymes responsible for
the organization of the photosynthetic apparatus. Therefore, the
changes in LHCB enzyme profiles (24–28 kDa) may have occurred as a
response to the imposed stress (reduction of luminous intensity) con-
ditions (wells 0–10, Fig. 2e and f).
4. Conclusion
Chlorella fusca LEB 111 supplemented with xylose and exposed to
6 h of light at an intensity of 28 μmolphotons m−2 s−1 induced the mi-
croalgal systems to produce 17.4% (w w−1) of PHB. The nitrogen re-
duction along with the pentose supply and 6 h of light stimulated
protein accumulation (53.2% w w−1). Reduction of light intensity and
addition of pentoses led to a reduction in the degradation of the enzyme
Rubisco. The results demonstrate that Chlorella fusca has the capacity to
accumulate PHB in high amounts through the metabolization of pen-
toses and the modifications in the light periodicity together with dif-
ferent luminous intensities.
462
Bioresource Technology 265 (2018) 456–463
Acknowledgements
The authors would like to thank the Coordination for the
Improvement of Higher Education Personnel (CAPES), the National
Council for Scientific and Technological Development (CNPq) and the
Ministry of Science, Technology, Innovations and Communication
(MCTIC) for the financial support provided.
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