Influence of nitrogen on growth, biomass composition, production, and properties of polyhydroxyalkanoates (PHAs) by microalgae

Accepted Manuscript Influence of nitrogen on growth, biomass composition, production, and properties of polyhydroxyalkanoates (PHAs) by microalgae

Samantha Serra Costa, Andréa Lobo Miranda, Bianca Bomfim Andrade, Denilson de Jesus Assis, Carolina Oliveira Souza, Michele Greque de Morais, Jorge Alberto Vieira Costa, Janice Izabel Druzian PII: DOI: Reference:

S0141-8130(18)31413-2 doi:10.1016/j.ijbiomac.2018.05.064 BIOMAC 9665

To appear in: Received date: Revised date: Accepted date:

26 March 2018 27 April 2018 12 May 2018

Please cite this article as: Samantha Serra Costa, Andréa Lobo Miranda, Bianca Bomfim Andrade, Denilson de Jesus Assis, Carolina Oliveira Souza, Michele Greque de Morais, Jorge Alberto Vieira Costa, Janice Izabel Druzian , Influence of nitrogen on growth, biomass composition, production, and properties of polyhydroxyalkanoates (PHAs) by microalgae. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.05.064

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ACCEPTED MANUSCRIPT Influence of nitrogen on growth, biomass composition, production, and properties of polyhydroxyalkanoates (PHAs) by microalgae Samantha Serra Costaa*, Andréa Lobo Mirandaa, Bianca Bomfim Andradeb, Denilson de Jesus Assisc, Carolina Oliveira Souzad, Michele Greque de Moraise, Jorge Alberto Vieira Costae, Janice Izabel Druziand Institute of Health Sciences, RENORBIO, Federal University of Bahia, Salvador,

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a

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Bahia, Brazil.

Institute of Health Sciences, Federal University of Bahia, Salvador, Bahia, Brazil.

c

Department of Chemical Engineering, Polytechnic School, Federal University of

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b

Bahia, Salvador, Bahia, Brazil.

Department of Bromatological Analysis, College of Pharmacy, Federal University of

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d

Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering,

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e

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Bahia, Salvador, Bahia, Brazil.

Federal University of Rio Grande, Rio Grande, Brazil.

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*Corresponding Author:

Me. Samantha Serra Costa

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Institute of Health Sciences Federal University of Bahia Av. Reitor Miguel Calmon, Canela 40231-300, Salvador, BA, Brazil Phone: +55 71 32836907 E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT This study sought to evaluate influence of nitrogen availability on cell growth, biomass composition, production, and the properties of polyhydroxyalkanoates during cultivation of microalgae Chlorella minutissima, Synechococcus subsalsus, and Spirulina sp. LEB-18. The cellular growth of microalgae reduced with the use of limited

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nitrogen medium, demonstrating that nitrogen deficiency interferes with the metabolism

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of microorganisms and the production of biomass. The biochemical composition of

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microalgae was also altered, which was most notable in the degradation of proteins and chlorophylls and the accumulation of carbonaceous storage molecules such as lipids and

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polyhydroxyalkanoates. Chlorella minutissima did not produce these polymers even in a nitrogen deficient environment. The largest accumulations of the

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polyhydroxyalkanoates occurred after a 15 days culture, with a concentration of 16% (dry cell weight) produced by the Synechococcus subsalsus strain and 12% by Spirulina

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sp. LEB-18. Polyhydroxyalkanoates produced by Synechococcus subsalsus and

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Spirulina sp. LEB-18 presented different thermal and physical properties, indicating the influence of producing strain on polyhydroxyalkanoates properties. The polymers

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obtained consisted of long chain monomers with 14 to 18 carbon atoms. This composition is novel, as it has not previously been found in PHAs obtained from

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Synechococcus subsalsus and Spirulina sp. LEB-18. Keywords: Synechococcus subsalsus; Chlorella minutissima; Spirulina sp., biopolymers.

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1. Introduction Microalgae establish a highly diverse group of photosynthetic microorganisms with key ecological importance and huge biotechnological potential, which has recently become a popular topic [1,2]. Microalgae, a group of microrganismsmicroorganisms with a

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short generation time, need some simple inorganic nutrients such as phosphate, nitrate,

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magnesium, and calcium as macro- and micronutrients for their growth and

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reproduction [3,4]. The microalgal biomass has high concentrations of lipids, proteins, and carbohydrates, which can be used for different applications.

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Microalgal biomass content of given target compounds, such as lipids, carbohydrates, pigments, is variable and can be modulated by altering cultivation conditions. The

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metabolic pathways of these microorganisms, when grown in deficient environments of one or more nutrients, are diverted, which may further the production and accumulation

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of various bioproducts. In this manner, the biochemical composition of microalgae can

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be manipulated by changing environmental conditions and environmental stresses, inducing the production of high concentrations of commercially important

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biocompounds such as polyhydroxyalkanoates [1]. Among the various nutrients in the medium, the carbon and nitrogen sources contribute more significantly to the overall

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cost of the production process and to the stimulation of the biopolymer synthesis. When the full potential of the microalgal biomass constituents is exploited, the byproducts of interest can be obtained simultaneously and the market value exceeds the production costs [5]. Polyhydroxyalkanoates (PHAs) are semi-crystalline polyesters synthesized and stored in microbial cells, the intracellular carbon and energy storage materials [1,3,6,7]. Owing to the renewability, degradability, almost CO2-neutral life cycle, and melting

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ACCEPTED MANUSCRIPT processability, polyhydroxyalkanoates are considered as good alternative had compoundgood alternatives for petroleum-derived synthetic plastics, and have immense applications in various fields such as food industry, agriculture, pharmaceuticals, and medicine. Polyhydroxyalkanoates also serve as the raw material for the production of pure chemicals in the paint industry [2,8]. Bacteria produces large amounts quantities of

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biopolymers in a short time. However, microalgae have the advantage of using smaller

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amounts of nutrients due to photosynthesis, which use the solar energy and transforms

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the carbon dioxide into oxygen, which is essential for humans. Thus, high growth rates and photosynthetic efficiency are critical qualities for microalgae employed for

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biopolymer production [1,9].

In addition to reducing production costs, it is necessary to improve the characteristics of

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the polymer to improve its processability and increase the market competitiveness of PHAs. Thus, it is fundamental to know the properties of the biopolymers obtained,

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verifying their possible industrial applications [10]. More than 150 monomers, formed

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from the hydroxyesters of fatty acids and comprised of carbon chains that may contain hundreds of carbons, can be combined within the PHA family to create polymers with

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extremely different properties for various applications [11]. The objective of this work was to evaluate the influence of nitrogen availability on cell

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growth, biomass composition, production, and the properties of polyhydroxyalkanoates obtained during cultivation of the microalgae Chlorella minutissima, Synechococcus subsalsus, and Spirulina sp. LEB-18. 2. Materials and Methods 2.1 Microorganism, culture media, and experimental conditions Chlorella minutissima and Synechococcus subsalsus were obtained from the Oceanographic Institute of the University of São Paulo (USP). Spirulina sp. LEB-18

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ACCEPTED MANUSCRIPT was obtained from the Culture Collection of the Laboratory of Biochemical Engineering at Federal University of Rio Grande (FURG). Cultures were performed in standard medium and limited nitrogen medium. The standard culture of Chlorella minutissima and Synechococcus subsalsus was performed with BG11 medium [12] containing mineral salts (K2HPO4, MgSO4, CaCl2, C6H11FeNO7, C10H14N2Na2O8, Na2CO3 and

of sodium nitrate (NaNO3) as the nitrogen source.

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C6H8O7), 0.40 g L-1 of sodium bicarbonate (NaHCO3) as the carbon source, and 1.5 g L-

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The standard culture of Spirulina sp. LEB-18 was carried out using Zarrouk medium containing mineral salts (K2HPO4, K2SO4, NaCl, MgSO4, CaCl2, FeSO4, and EDTA),

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16.8 g L-1 of sodium bicarbonate (NaHCO3) as the carbon source, and 2.5 g L-1 of sodium nitrate (NaNO3) as the nitrogen source [13]. The cultures in the limited medium

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were carried out using the respective standard medium with an approximately 70% reduction of the nitrogen source, that is, sodium nitrate (NaNO3).

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Experiments were performed in duplicate in an acrylic cylindrical photobioreactor, with

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a working volume of 20 L, 450 mm height, and 250 mm of diameter. The cultures were maintained for 20 days, from an initial cell concentration of approximately 0.2 g L-1, at

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28 °C in a growth chamber under a 12 h light / dark photoperiod. The illumination was provided with 40 W daylight-type fluorescent tubular lamps that produced an

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illuminance of 41.6 μmolphotons m-2 s-1 [13]. The evaporated water was replenished every two days, prior to sampling, with sterile water. Stirring was effected by compressed air injection at 0.3 vvm. To avoid contamination, the pumped air was filtered by glass wool filters coupled to the system. Each experiment consisted of three replicates. 2.2 Analytical determinations

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ACCEPTED MANUSCRIPT Cell growth was monitored every two days by optical density using a digital spectrophotometer (Bel Photonics UV-MS1) at a wavelength of 570 nm, 630 nm, or 670 nm for Chlorella minutissima, Synechococcus subsalsus, and Spirulina sp. LEB-18, respectively. Prior to the experiments, a standard growth curve of each strain, in standard medium and in limited medium, was generated to correlate the optical density

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with the dry weight biomass as indicated in Table 1.

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The pH measurements were performed on cultures every two days using a digital pH

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meter (SANXIM PHS-3D). The chlorophyll content was measured every two days using 1 mL of liquid culture. The samples were centrifuged at 2000 ×g for 5 min

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(Eppendorf® 5702-R), and the pellet was resuspended in methanol. Chlorophyll was extracted with a 24 hr incubation at 4 °C (in the dark), and absorbance was measured at

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652.4 and 664.2 nm as previously described by Freitas et al. [14].

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Chlorella minutissima

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Synechococcus subsalsus

Spirulina sp. LEB-18

Growing medium

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Microalgae strain

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Table 1. Standard growth curves for each strain in standard or limited medium. Standard growth curve

Standard medium

y = 0.5045x - 0.091, R2=0.9926

Limited medium

y = 0.527x – 0.0277, R2= 0.9911

Standard medium

y = 1.3964x – 0.0077, R2=0.9909

Limited medium

y = 1.5527x – 0.0169, R2=0.9919

Standard medium

y = 0.6170x – 0.0125, R2=0.9916

Limited medium

y = 1.438x – 0.0092, R2=0.9901

After 20 days of cultivation, the total biomass from each experiment was recovered by centrifugation (Hitachi Himac CR-GIII, Tokyo-Japan) at 10,000 ×g for 15 min, resuspended in distilled water, and centrifuged again under the same conditions to

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ACCEPTED MANUSCRIPT improve nutrient removal. The biomass was frozen at -80 °C, lyophilized, and stored at -20 °C until characterization. 2.3 Growth parameters 2.3.1 Biomass productivity The biomass productivity was calculated as shown in Eq. (1), where Xt is the biomass

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concentration (g L-1) at time t (d), and X0 is the biomass concentration (g L-1) at time

𝑋𝑡 −𝑋0 𝑡−𝑡0

Eq. (1)

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𝑃𝑥 =

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t0(d).

2.3.2 Maximum specific growth rate

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The maximum specific growth rate was calculated via linear regression applied to the

t(d). 2.4 Characterization of biomass

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logarithmic growth rate of each experiment obtained from a plot of ln X (g L-1) versus

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2.4.1 Quantification of proteins and lipids The protein concentration was determined using the Kjeldahl method [15], with a conversion factor of 5.22, which is specific for microalgae [16]. The total lipid content

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of the biomass was extracted with chloroform:methanol (2:1) and quantified by

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gravimetry as described by Folch et al. [17]. 2.4.2 Microalgae fatty acid composition The individual fatty acids were identified by gas chromatography. Aliquots of fat were saponified, followed by methylation. Fatty acid methyl esters (FAME) were extracted and stored in an inert atmosphere (N2) at −60 °C. Methyl tricosanoate 23:0 (T9900; Sigma Aldrich®), was added as an internal standard [8]. The FAME were separated and identified on a gas chromatograph (Clarus 680; Perkin Elmer®) with a DB-FFAP column (30 m × 0.32 mm × 0.25 mm) and equipped with a flame ionization detector. 7

ACCEPTED MANUSCRIPT The analysis parameters included an injector temperature of 250 °C and detector temperature of 280 °C. The following thermal cycle was used: 150 °C for 16 min, then increased by 2 °C min-1 until the temperature reached 180 °C, maintained for 25 min, increased by 5 °C min-1 until the temperature reached 210 °C, and maintained for 25 min. Helium was used as a carrier gas at 1.0 mL min−1. Hydrogen gas and synthetic air

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flows were provided at 30 and 300 mL min−1, respectively. The injections were

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performed in duplicate for each extraction in a 1 μL volume. FAME were identified by

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comparing retention times with known mix standards (189–19; Sigma-Aldrich®). The quantification of fatty acids, expressed in milligrams per gram of lipids, was performed

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by the addition of an internal standard (C23:0). All analyses were conducted in duplicate. To ensure the conclusive identification of fatty acids, the samples were

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injected in a gas chromatograph coupled to a gas chromatography mass spectrophotometer (Clarus 500; Perkin Elmer®). The mass fragments of the samples

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were compared with spectral data from the National Institute of Standards and

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Technology (NIST) standard mass spectral databases and mix standards under the same operating conditions used in the GC/FID at a spectrum of 50–500 m/z (EI, 70 eV).

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2.4.3 Extraction, quantification of PHAs Extraction of polyhydroxyalkanoates was performed as previously described by Yellore

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and Desia [18], with certain modifications. Every five days, a 1 L sample was withdrawn from the cultures to determine the polyhydroxyalkanoates content produced. A known quantity of microalgae cells were combined with sodium hypochlorite (10.0%, v v-1) and distilled water to give a final concentration of 4.0% (v v-1) that was incubated for 20 min at 45 °C. The sample was then centrifuged at 15,700 × g for 20 min, and the polymer was extracted in hot chloroform followed by precipitation with cold methanol.

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ACCEPTED MANUSCRIPT The sample was then centrifuged at 15,700 × g for 20 min to obtain a pellet. The pellet was dissolved again in hot chloroform and dried at 60 °C. Biomass production and polyhydroxyalkanoates (obtained after extraction) were calculated using a gravimetric method, and expressed as g L−1. The polyhydroxyalkanoate extraction yield was calculated by the ratio of the

𝑚𝑝 𝑋 100

Eq. (2)

𝑚𝑏

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𝑌=

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polyhydroxyalkanoate concentration to the biomass concentration according to Eq. (2).

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Where,

Y is the extraction yield of polyhydroxyalkanoates in percentage,

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mp is the mass of polyhydroxyalkanoates obtained in grams, and mb is the mass of the biomass used in the extraction in grams.

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2.4.4 Thermogravimetric Analysis (TGA)

Thermogravimetric analysis (TGA) (PerkinElmer Model Pyris 1 TGA Waltham,

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Massachusetts, USA) of biomass was conducted to determine the initial degradation

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temperature (Tonset) and the maximum decomposition temperature (Tdecomp). Biomass (5 mg) was folded into a platinum tray and heated at a rate of 10 °C min-1, to increase the

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temperature from 30 to 700 °C, under a nitrogen flow rate of 20 mL min−1 [7].

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2.5 PHAs characterization 2.5.1 Fourier Transform Infrared Spectroscopy (FTIR) Polyhydroxyalkanoates samples were analyzed qualitatively by FTIR spectroscopy (Perkin Elmer Model Spectrum 100, Perkin Elmer, Waltham, Mass., USA) between 4000 cm−1 and 600 cm−1 using a single-bounce ATR accessory with a Zinc selenide crystal [7]. 2.5.3 Molecular weight (Mw)

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ACCEPTED MANUSCRIPT The Mw of polyhydroxyalkanoates were obtained by size-exclusion chromatography with HPLC (PerkinElmer Series 200) equipped with an autosampler and a refractive index (RI) detector (PerkinElmer), as previously described by Assis et al. [19]. For polyhydroxyalkanoates separation, Shodex KD 807 column (30 cm × 78 mm × 5 mm) was employed at 30 °C. The polymers were dissolved in chloroform to a final

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concentration of 0.7 g L−1, and filtered (PTFE membrane, 0.45 μm), before separation.

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Chloroform was used as the mobile phase, at a flow rate of 1.0 mL min−1. A standard

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curve, Eq. (3) was created using low polydispersity polystyrene standards (682– 1.670.000 Da) (Polystyrene High Mw Standards Kit, Polymer Standards Service, USA). Eq. (3)

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𝑙𝑜𝑔 𝑀𝑤 = −0.8364 𝑥 𝑇𝑟 + 14.83 with R2 = 0.9917 Where Tr is the retention time.

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2.5.3 Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC)

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Thermogravimetric analysis (TGA) (PerkinElmer Model Pyris 1 TGA Waltham,

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Massachusetts, USA) of PHAs was conducted to determine the initial degradation temperature (Tonset) and the maximum decomposition temperature (Tdecomp).

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Polyhydroxyalkanoates (5 mg) were folded into a platinum tray and heated at a rate of 10 °C min-1, to increase the temperature from 30 to 600 °C, under a nitrogen flow rate

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of 20 mL min−1 [19]. The melting temperature (Tm) and crystallinity of PHAs were determined by differential scanning calorimetry (DSC) (SHIMADZU DSC-50). Polyhydroxyalkanoates (2 mg) were encapsulated in an aluminum pan and heated from 25 to 600 °C at a heating rate of 10 °C min-1, with liquid nitrogen used as the coolant and helium as the purge gas. The Tm and enthalpy of fusion (∆Hm) were determined from the melting endotherm and the glass transition temperature (Tg). The ∆Hm of a theoretical 100% crystalline (∆Hm100%) sample was assumed to be 146 J g−1. The

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ACCEPTED MANUSCRIPT crystallinity of polyhydroxyalkanoates by DSC (% Xc) was calculated using the ratio of ∆Hm100%–∆Hm [10] according to Eq. (4). ∆𝐻𝑚

% 𝑋𝑐 = ∆𝐻

Eq. (4)

𝑚100%

2.5.4 Monomeric composition PHAS

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The quantity and composition of the constituent polyhydroxyalkanoates monomers were determined using Gas Chromatography–Mass Spectrometry (GC–MS; Clarus 500

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PerkinElmer) with the TurboMass software version 4.5.0 and the NIST 98 library.

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Approximately 4 mg of the produced dry polyhydroxyalkanoates was subjected to methanolysis as previously described by Brandl et al. [20], but with the modifications

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proposed by Campos et al. [7]. A portion of the organic phase was separated after the splitless injection on a capillary column DB-1 (30 m × 0.25 mm × 0.25 mm). Helium

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(flow 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

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scan between 50 and 550 m/z. The following temperature program was applied: 80–200

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°C (20 °C min-1). The mass spectra were compared with the spectra from the 98 NIST library, and alkanoates monomers were quantified by area normalization.

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2.6 Statistical analysis

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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 Growth and characterization of biomass Figure 1 shows biomass production during the cultivation of the microalgae Chlorella minutissima, Synechococcus subsalsus, and Spirulina sp. LEB-18. For the three strains, the cultures in standard medium and in limited nitrate medium did not present the lag 11

ACCEPTED MANUSCRIPT phase of adaptation, instead beginning in the exponential phase, because the inocula were adapted to the culture media used. Cell growth of the microalgae in the standard medium and the limited nitrate medium was similar for the first 4 days of Chlorella minutissima culture, 6 days of Synechococcus subsalsus culture, and 10 days of Spirulina sp. LEB-18 culture (Figure 1). After these periods, cell growth stopped or

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began to decline for the three strains, demonstrating that nitrogen deficiency in the

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culture media interferes directly with the metabolism, and biomass production, of these

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microorganisms.

Moreover, a 20% reduction in the maximum productivity of the Chlorella minutissima

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culture was observed, which went from 0.025 g L-1 d-1 when standard medium was used to 0.020 g L-1 d-1 when limited medium was used (Table 2). A similar behavior was

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observed for the specific growth rate (μmax), which showed a reduction of 23% when nitrogen was limited (Table 2).

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With regard to Synechococcus subsalsus, there was a reduction of approximately 93%

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in maximum productivity and 39% for μmax when the limited nitrogen medium was used (Table 2). For Spirulina sp. LEB-18, the reduction was approximately 11% for Pmax and

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26% for μmax when the nitrogen in the medium was limited.

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Table 2. Maximum biomass productivity and specific growth rate of cultures.

Microalgae strain

Chlorella minutissima Synechococcus subsalsus Spirulina sp. LEB-18

Growing medium SM LM SM LM SM LM

Maximum biomass productivity (Pmax, g L-1 d-1) 0.025 0.020 0.145 0.009 0.018 0.016

Specific growth rate (μx, d-1) 0.061 0.047 0.039 0.024 0.043 0.032

SM is standard medium and LM is limited medium.

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ACCEPTED MANUSCRIPT The nitrate consumption during cultivation varied greatly when comparing the nitrate concentration reductions in standard medium and in limited medium. For Chlorella minutissima there was a 0.29 g L-1 reduction in nitrate concentration after 20 days of culture in standard medium, while the reduction concentration in a limited medium was 0.25 g L-1 (Figure 1A). These data suggest that nitrogen consumption occurs at a higher

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rate in the standard medium than the limited medium. For Synechococcus subsalsus and

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Spirulina sp. LEB-18, the difference in consumption between standard and limited media was even greater. For Synechococcus subsalsus, the nitrate concentration in the

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medium after 20 days of cultivation was 0.48 g L-1 in standard medium and 0.22 g L-1 in

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limited medium (Figure 1B). For Spirulina sp. LEB-18, the concentration of nitrate in the medium after 20 days of cultivation was 0.65 g L-1 in standard medium and 0.25 g

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L-1 in limited medium (Figure 1C). These data point to a lower nitrogen consumption rate in the cultures carried out in a limited nitrate medium, which were responsible for

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the changes in the metabolism of these microalgae.

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The high nitrogen rate contributes to growth, and therefore its consumption was also responsible for improving growth. Nitrogen is quantitatively the second most important

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inorganic nutrient for microalgae, and is available in the culture media employed in this study through sodium nitrate. Nitrate, which is absorbed by microalgae cells via an

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active transport system, is reduced to nitrite by nitrate reductase, and then to ammonium by nitrite reductase [21]. Furthermore, when nitrate is the nitrogen source for growth, there is a significant increase in the demand for reducers in competition with photosynthetic CO2 fixation [22]. These results are consistent with the previous literature, where it is well documented that nitrogen limitation of microalgae decreases microalgal growth and biomass production while altering biomass biochemical composition [23]. The μmax reduction

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ACCEPTED MANUSCRIPT indicates changes in the metabolic pathways of these microalgae, since they are known to be actively growing cells where energy is diverted to the synthesis of storage compounds, including biopolymers, so that lower μmax values occur. The pH of the three microalgae cultures presented discrete variations over time, ranging from 8.6 to 9.8 for Chlorella minutissima, 9.0 to 9.8 for Synechococcus subsalsus, and

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9.2 to 9.8 for Spirulina sp. LEB-18. No variation in pH was observed between cultures

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made in standard medium and in limited nitrogen medium for the microalgae

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Synechococcus subsalsus or Spirulina sp. LEB-18. A greater variation was found for the Chlorella minutissima cultures, where the culture in standard medium presented a pH

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between 9.0 and 9.8 and the culture in limited nitrogen medium had a pH between 8.6 and 9.5. The pH is one of the most critical environmental conditions in microalgal

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cultivation, since it determines the solubility and availability of CO2 and nutrients and has a significant influence on microalgal metabolism [24]. Each microalgal species has

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an optimal pH range for biomass and lipid production, which is narrow and strain

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specific [25].

According to Vonshak [26], the optimum pH for microalgae ranges from pH 8.5 to

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10.5, with a reduction in cell numbers occurring at pH 8.0 and below. Jiménez et al. [27] report that pH 9.5 and above is ideal for cultivation of Spirulina, with their

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Spirulina cultures in Malaga having pH values of pH 9.0 to 10.9, similar to the pH 9.2 to 9.8 observed in our study. For Chlorella, Qiu et al. [28] states that a pH between 7.0 and 8.0 is ideal for growth of this microalga, and cultivation outside this range may compromise its growth. This may explain the low cell concentration (Figure 1) found in the Chlorella minutissima cultures used in this study. According to Cuaresma et al. [29], an increase in pH in photosynthetic cultures occurs because of the biological activity of the cells, which reduces the dissolved inorganic carbon content because of the

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ACCEPTED MANUSCRIPT consumption during cell growth. Thus, there exists a displacement of the carbonatebicarbonate equilibrium in the buffer system. The chlorophyll concentration, of the cultures of the three microalgae, presented a similar pattern as the cell growth (Figure 2). This was expected since the production of chlorophyll by microalgae is part of its primary metabolism, and thus is directly related

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to the production of biomass. The highest concentrations of chlorophyll for the three

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microalgae were found using standard medium. Chlorella minutissima showed the highest chlorophyll concentration (2.9 μg mL-1 of culture) at 6 days of culture in

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standard medium, while the highest levels found for Synechococcus subsalsus (2.4 μg

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mL-1 culture) and Spirulina sp. LEB-18 (1.9 μg mL-1 culture) were reached at the end of the cultures in standard medium. By limiting the availability of nitrogen in the culture

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media, chlorophyll concentrations dropped after 4, 7, and 5 days for Chlorella minutissima, Synechococcus subsalsus, and Spirulina sp. LEB-18, respectively (Figure

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2). During nitrogen starvation, the cells gradually change from a vegetative state to a

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dormant state. The most obvious feature of this was the change in color from blue-green to brownish-yellow, which occurred for all microalgae. This phenomenon is called

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"nitrogen chlorosis" and is caused by the degradation of the pigments phycocyanin and chlorophyll [11].

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In the work carried out by Gorl et al. [30], when transferring Synechococcus PCC7942 to a nitrogen depleted medium, 95% phycocyanin was degraded within 24 h. Additionally, after 10 days 95% of the chlorophyll was also degraded. According to Markou et al. [31], when microalgae are deprived of nitrogen the synthesis of biomolecules rich in nitrogen (proteins, chlorophylls) is reduced and biomolecules rich in carbon (carbohydrates and/or lipids) are accumulated. In this manner, nutrient limitation, particularly nitrogen, is often an effective strategy to increase specific target

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ACCEPTED MANUSCRIPT compounds in the biomass such as accumulation of lipids, carbohydrates, and carotenoids. According to Adams et al. [23], even though nitrogen limitation results in an accumulation of target compounds, biomass growth rate declines significantly. A declined growth rate results in an overall decrease of the productivity of the target

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compound, as observed in this study. Moreover, it is well documented that nitrogen

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limitation results in a decrease in microalgal biomass protein content [32], particularly

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in proteins associated with the photosynthetic apparatus [31].

In this study, the most notable change in biochemical composition is the degradation of

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proteins and photosynthetic pigments (chlorophylls), and the accumulation of carbonaceous storage molecules such as lipids and polyhydroxyalkanoates. Lipid and

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protein analysis showed that nitrogen limitation caused lipid accumulation and a reduction of protein content at the end of the culture (Table 3). There was a 15%

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reduction in the protein content of Chlorella minutissima biomass, and a 40% increase

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in lipid content, when limiting the availability of nitrogen in the culture medium (Table

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3).

Table 3. Determination of protein, lipids, and polyhydroxyalkanoates in the microalgal

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biomass after 20 days of cultivation. Microalgae strain Chlorella minutissima

Synechococcus subsalsus Spirulina sp. LEB-18

Growing medium SM LM SM LM SM LM

Proteins (%) 48.71 ± 1.13a 41.34 ± 1.22b 59.22 ± 0.87c 50.03 ± 1.23a 57.70 ± 0.75d

Lipids (%) 13.21 ± 0.12ª 18.54 ± 0.89b 10.11 ± 0.33c 12.96 ± 1.02ª 11.40 ± 0.52d

48.24 ± 0.44a

13.67 ± 1.13a

PHAs (%) 2.16 ± 0.63a 7.87 ± 0.71b 2.02 ± 0.40a 9.56 ± 0.76c

Means ± standard deviations. The equal letters in the same column indicate that there is no significant difference between the experiments performed at 95% confidence level (p < 0.05). SM is standard medium and LM is limited medium.

16

ACCEPTED MANUSCRIPT

For Synechococcus subsalsus, the protein reduction was 16% and the increase in lipid content was 28%. For Spirulina sp. LEB-18, protein reduction was 14% and the increase in lipid concentration in the biomass was approximately 20% when using the limited medium (Table 3). These observations are generally in line with previously

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published studies, which report that the degree of nutrient limitation proportionally

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affects the microalgal biochemical composition and growth [23].

SC

In addition to accumulating lipids with the reduction of nitrogen availability during cultivation, the microalgae Synechococcus subsalsus and Spirulina sp. LEB-18 also

NU

increased the accumulation of polyhydroxyalkanoates (Table 3, Figure 3). The largest polymer accumulations occurred at 15 days of cultures, where a concentration of

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approximately 16% was found for Synechococcus subsalsus strain and 12% for Spirulina sp. LEB-18 (Figure 3). Decreasing the amount of nitrogen in the culture

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medium resulted in a 400% increase in synthesis of the polymer by Synechococcus

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subsalsus and a 300% increase by Spirulina sp. LEB-18. By means ofBased on these results, it is evident that the nitrogen reduction of the availability of nitrogen in the

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culture media is an effective tool to stimulate the production of PHAs by these microalgae. Chlorella minutissima was not able to accumulate the polymer, even in a

AC

nitrogen deficient environment. According to Kavitha et al. [1], Chlorella sp. is a eukaryotic organism of the green algae genus, which accumulate large amounts of carbon, mostly in the form of lipids. Thus, the maximization of PHA production in green algae will likely require that the carbon normally used for synthesis of storage lipids or starch synthesis be diverted to PHA synthesis. Thus, the limitation of nitrogen in the culture medium alone is not able to stimulate the synthesis of PHAs by these organisms, necessitating a more detailed knowledge of the mechanisms involved in

17

ACCEPTED MANUSCRIPT controlling carbon flux through various metabolic pathways essential to achieve this goal. Poirier et al. [33] state that it is likely that the expression of a number of genes will have to be involved in regulating the activity of various metabolic pathways that lead to PHA production by eukaryotic organisms. The increased accumulation of polyhydroxyalkanoates by the microalgae

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Synechococcus subsalsus and Spirulina sp. LEB-18 started at the end of the exponential

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phase of cell growth and beginning of the stationary phase, when microbial growth

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ceases (Figure 1). Biopolymers, such as polyhydroxyalkanoates, serve as intracellular energy reserves, because their production generally occurs during the exponential phase,

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in parallel with whatever factor is used to measure cell growth. Here cell growth was assessed in term of increases in biomass [9,32]. This aids in the survival of the

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reproducing microorganism, which can use polyhydroxyalkanoates for survival in the later stages of growth when nutrients become limiting. This justifies the reduction of

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already beginning (Figure 3).

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polyhydroxyalkanoates content at the end of culture, when the period of cell death was

In the case of cyanobacteria, Synechococcus subsalsus and Spirulina sp. LEB-18 shows

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that both are rich sources of proteins (Table 3). These data imply a large nitrogen requirement for growth. Therefore, under nitrogen limitation, they divert carbon into

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other metabolic routes, and produce biopolymers to serve the carbon and energy storage compounds, which can be reused when conditions become more favorable. When the Favorable conditions include periods where the nitrogen content of the environment increases, and the organism can produce proteins for cell growth rather than the storage lipids from which polyhydroxyalkanoates derives. Thus, these results demonstrate that the amount of nitrogen available is known to directly influence biopolymer synthesis. Increased PHA production of PHAs by other cyanobacteria subjected to stress

18

ACCEPTED MANUSCRIPT conditions due to , because of phosphorus and/or nitrogen limitation, has already been highlighted in otherprevious studies [9,23]. However, lower percentages of accumulation percentages were obtained than those obtained in this study, which showsindicates a potential offor obtaining these biopolymers byfrom Synechococcus subsalsus and Spirulina sp. LEB-18.

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The results obtained from the thermal analysis of the samples were consistent with the

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biochemical composition found for the biomass. The derivative thermogravimetric

SC

(DTG) data demonstrated the existence of three stages of mass loss characteristic of the microalgae biomass (Figure 4). The first stage begins at approximately 30 °C and ends

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near 150 °C, this is related to the loss of water from the biomass. Thus, the mass loss in this stage depends strongly on the moisture content of the sample. The second stage is

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the most significant and occurs between 250 and 450 °C. In this stage, all microalgal components (carbohydrates, proteins, lipids, and other minor components) are

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decomposed to produce volatile chars and releases. The third stage starts at 500 °C, and

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shows a slight mass loss due to the degradation of carbonaceous matters in the solid residues [34].

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As the biomass of microalgae is composed mostly of proteins and lipids, it is observed that, for the three microalgae, the greatest mass loss occurred in the second stage of

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decomposition. As mentioned, these stage triggers are because of the thermal decomposition of carbohydrates and proteins [35], and the thermal degradation of carbohydrates and proteins normally coincides. The decomposition peak was accompanied by smaller peaks, which are a consequence of thermal lipid degradation. This arises from the higher decomposition temperature of lipids compared to that of carbohydrates or proteins [36]. Thus, when nitrogen was limited in culture medium, there was an increase in lipid contents, and consequently an increase of the shoulder

19

ACCEPTED MANUSCRIPT formed after the most expressive peak of this stage. This can be observed mainly in the cultures of Chlorella minutissima, where there was a more significant increase in the lipid content of the biomass (Figure 4A). Table 4 presents the values of Tonset, Tdecomp, and weight loss of the biomass cultures. In Synechococcus subsalsus and Spirulina sp. LEB-18 cultures, a Tonset reduction at the

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second stage of biomass decomposition was obtained using a limited nitrogen medium.

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This is due to the increase in the content of polyhydroxyalkanoates in the biomass,

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which begins decomposition near 260 °C [7] and causes reduction of Tonset for the main stage.

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Table 5 shows the concentrations of fatty acids for the assays performed in standard medium and in limited medium with the three microalgae. For Chlorella minutissima

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and Synechococcus subsalsus a higher concentration of stearic acid (C18:0) was observed in the biomass obtained from the two-culture media, with a high content of

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saturated compounds. For Spirulina sp. LEB-18, a higher concentration of palmitic acid

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(C16:0) was observed. With the use of limited medium, fatty acid profiles obtained did not change, however, changes in fatty acid concentrations occurred. According to

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Sakarika and Kornaros [37], the fatty acid profile is a characteristic fingerprint for a

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certain microalga at a certain cultivation mode.

Table 4. Values of Tonset, Tdecomp, and weight loss of the culture biomass. 1st stage Microalgae strain

Growing medium

Chlorella minutissima Synechococcus subsalsus Spirulina sp. LEB-18

2nd stage

3rd stage

Tonset (°C)

T decomp. (°C)

51.18

weight loss (%) 7.48

Tonset (°C)

T decomp. (°C)

322.23

weight loss (%) 34.82

506.37

509.99

weight loss (%) 6.42

255.72

27.18

53.99

12.67

290.46

351.63

43.70

364.35

488.94

11.83

SM

31.39

57.82

8.79

308.40

365.88

39.50

435.90

503.59

11.44

LM

25.65

57.46

14.01

296.85

369.89

55.59

476.82

500.45

8.55

SM

42.70

62.51

11.67

285.07

322.76

48.48

590.13

623.23

23.00

LM

37.34

63.10

8.47

278.39

325.49

51.42

551.84

615.02

30.01

Tonset (°C)

T decomp. (°C)

SM

28.60

LM

20

ACCEPTED MANUSCRIPT SM is standard medium and LM is limited medium.

For Chlorella minutissima, the use of the limited medium caused an increase in the concentration of the fatty acids, which changed from 52.86 mg g-1 to 82.59 mg g-1 as a result of the higher lipid production (Table 5). The saturated and unsaturated fatty acid

PT

contents also increased when the limited medium was used to culture of this microalga.

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For Synechococcus subsalsus and Spirulina sp. LEB-18, a reduction in the concentration of the total fatty acids, as well as of the unsaturated fatty acids. , was

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observed.

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As the microalgae Synechococcus subsalsus and Spirulina sp. LEB-18 increased the accumulation of polyhydroxyalkanoates in limited nitrogen media, part of the lipids

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formed were converted into these biopolymers. Hu et al. [38] thoroughly explained the mechanisms of new lipid synthesis, indicating that saturated fatty acids are synthesized

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first and double bonds are introduced later by the soluble enzyme stearoyl-acyl

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desaturase to form polyhydroxyalkanoates. According to Sakarika and Kornaros [37], nitrogen limitation not only enhances the lipid productivity, but also improves the lipid

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profile for the production of bioproducts such as biopolymers and biodiesel, reduces the

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requirement for nitrogen addition, and results in cost and energy savings.

Table 5. Fatty acid composition of microalgae biomass after 20 days of culture.

Fatty acids C4:0 C6:0 C8:0 C10:0 C11:0 C13:0 C14:0

Concentration (mg g-1 of lipid)

Chlorella minutissima SM LM 0.14 ± 0.03 0.20 ± 0.07 0.30 ± 0.05 2.50 ± 0.18 2.07 ± 0.03

0.26 ± 0.08 0.31 ± 0.01 0.27 ± 0.12 1.04 ± 0.10 3.14 ± 0.26 2.85 ± 0.60

Synechococcus subsalsus SM LM 0.24 ± 0.06 0.27 ± 0.04 0.14 ± 0.01 0.61 ± 0.03 5.37 ± 0.01 5.03 ± 0.53

0.48 ± 0.06 0.48 ± 0.01 0.23 ± 0.08 0.37 ± 0.05 4.57 ± 0.03 4.33 ± 0.04

Spirulina sp. LEB-18 SM LM 2.91 ± 0.44 0.29 ± 0.03 0.14 ± 0.02 0.87 ± 0.06 0.28 ± 0.04 3.45 ± 0.24 1.44 ± 0.23

1.75 ± 0.08 0.36 ± 0.01 0.15 ± 0.02 1.21 ± 0.08 0.16 ± 0.05 2.97 ± 0.14 1.27 ± 0.12 21

ACCEPTED MANUSCRIPT 2.26 ± 0.25 28.12 ± 1.80 0.52 ± 0.08 8.94 ± 0.09 13.94 ± 0.11 3.91 ± 0.19 45.45 ± 2.34 20.11 ± 1.56 60.56 ± 3.10

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C14:1w5 3.25 ± 0.82 6.78 ± 0.02 8.69 ± 0.22 6.19 ± 0.21 0.28 ± 0.05 C16:0 5.91 ± 0.06 6.39 ± 0.48 9.11 ± 0.41 13.33 ± 0.27 18.27 ± 1.09 C17:0 1.43 ± 0.01 3.38 ± 0.02 1.03 ± 0.07 2.71 ± 0.65 0.48 ± 0.10 C18:0 30.73 ± 0.27 51.42 ± 0.36 12.65 ± 0.44 16.58 ± 0.25 12.20 ± 0.07 C18:1w9 6.32 ± 0.52 6.75 ± 0.66 15.91 ± 0.35 4.39 ± 0.26 14.92 ± 0.01 C18:2w6 11.77 ± 1.25 C18:3w6 3.90 ± 0.11 Σ saturated 43.29 ± 0.98 69.07 ± 1.19 34.44 ± 0.82 43.07 ± 1.16 40.33 ± 1.98 Σ unsaturated 9.57 ± 0.67 13.52 ± 0.87 24.60 ± 1.10 10.59 ± 0.91 30.87 ± 1.52 Total 52.86 ± 1.13 82.59 ± 1.46 59.04 ± 1.63 53.66 ± 1.83 71.20 ± 4.66 Means ± standard deviations. SM is standard medium and LM is limited medium.

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3.2 PHAs characterization 3.2.1 FTIR spectra

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Figure 5 shows the FTIR spectra of the PHA samples produced by two strains of

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cyanobacteria, showing a similarity between the samples. The main bands observed in the PHA spectrum were assigned to the axial deformation of the ester carbonyl group (C=O) (1710-1750 cm -1) and the formation of the C-O-C groups appeared in the

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spectral region from 1260 to 1300 cm -1 (crystalline phase) and about 1050 cm -1 [39].

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The bands close to 1380 cm -1 are associated with symmetric angular deformation in the plane of the methyl groups (CH3), and the bands close to 980 cm -1 correspond to the

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vibration of the ester group carbonyl (C-C) [10]. These functional groups are

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characteristic of the PHA chemical structures, confirming the production of these biopolymers by the studied microalgae. 3.2.2 PHA molecular weight The Mw for the PHA produced by the microalgae Synechococcus subsalsus was 179.66 kDa, whereas the Mw found for the PHA obtained from Spirulina sp. LEB-18 was 163.26 kDa (Table 6). These data demonstrate that the Mw of the polymer varies based on the characteristics of the producing strain. This difference in behavior, observed in the polymers obtained in this study, is most likely attributable to a structural 22

ACCEPTED MANUSCRIPT rearrangement of the polymer during biosynthesis of each strain [7]. This structural rearrangement may have been caused by increased degree of polymerization, which formed longer chains with different MW. Laycock et al. [40] suggested that Mw distribution is associated with the end-use properties of the biopolymers via the structural control of macromolecules.

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PHA properties depend on the size of the polymer chains, whose structural

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rearrangements may depend on the degree of polymerization [19]. Thus, as the

SC

polymers obtained by the two microalgae presented different Mw, they consequently present different properties (physical, mechanical, thermal, rheological, and others).

NU

Owing to the significant industrial competition, the ability to control the Mw of the polymer during its production, and understanding how Mw influences the final

MA

properties of the polymer, are extremely important.

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Table 6. Mw and thermophysical properties of polyhydroxyalkanoates produced by

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cyanobacteria Synechococcus subsalsus and Spirulina sp. LEB-18. PHA producer strains

Mw (kDa)

Spirulina sp. LEB-18

179.66 ± 1.75a *

163.26 ± 7.94b *

286.88

249.79

AC

Tonset (°C)

CE

Synechococcus subsalsus

Tdecom (°C)

312.19

269.76

Tm (◦C)

173.53

171.69

∆Hm (J g-1)

54.15

65.93

Xc (%)

37.09

45.15

Mw signifysignifies molecular weight; Tonset signifysignifies initial decomposition temperature; Tdecom signifysignifies maximum decomposition temperature; Tm signifysignifies melting temperature; ∆Hm signifysignifies melting enthalpy; Xc signifysignifies degree of crystallinity.

23

ACCEPTED MANUSCRIPT *Means ± standard deviations. *The equal letters in the same line indicate that there is no significant difference between the experiments performed at 95% confidence level (p < 0.05).

3.2.3 Thermal characterization Table 6 shows the thermophysical properties of PHAs. Figure 6A shows the associated

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derivative thermogravimetric curves. The initial decomposition temperature (Tonset) of PHA was 286.88 °C and 249.79 °C for samples produced by Synechococcus subsalsus

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and Spirulina sp. LEB-18, respectively, demonstrating that the polymers obtained can

SC

be thermally stable at temperatures below 240 °C. Thermal degradation occurred during

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the first stage of weight loss, where 96.74% of the PHA produced by Synechococcus subsalsus and 93.49% for PHA produced by Spirulina sp. LEB-18 was degraded. This

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behavior is typical of PHA samples [7,10], and indicated the presence of remaining impurities from the PHA extraction process [19]. Thus, these results suggest that the

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PHA obtained from the microalgae Synechococcus subsalsus produces a higher

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percentage of pure polymer when compared to that obtained from Spirulina sp. LEB-18. The Tm, melting enthalpy (∆Hm), and degree of crystallinity (Xc) are listed in Table 6.

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Differential scanning calorimetry (DSC) curves of the obtained PHA (Figure 6B) showed endothermic peaks higher than 170 °C (Tm) associated with crystalline melting

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of these biopolymers. These results are similar to those found by Bhati and Mallick [41], who analyzed PHA produced by the cyanobacteria Nostoc muscorum and found Tm values close to 178 °C. Alternately, Hermann-Krauss et al. [42] have obtained PHAs produced by bacteria Burkholderia cepacia at lower melting temperatures (130 140 °C) than that obtained in this study. According to Pohlmann et al. [43], PHA copolymers are unstable above their melting temperatures, which renders them susceptible to molecular degradation and causes difficulties in the processing of

24

ACCEPTED MANUSCRIPT bioplastics. Thus, the two polymers can be easily molded by injection and are applicable for blown film processing [44]. Based on ΔHm, we note that the degrees of PHA crystallinity was 37.09% for samples produced by Synechococcus subsalsus, and 45.15% for those produced by Spirulina sp. LEB-18. PHAs are semicrystalline polymers, and their degree of crystallinity depends

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directly on their composition. According to Assis et al. [19], PHAs with crystallinity

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between 60 and 80% are considered rigid. Flexible and more elastic PHAs have

SC

medium - (30–40%) and short (< 30%) - chain polymer lengths respectively. The lower degree of crystallinity increases the number of possible industrial PHA applications by

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improving its processing characteristics, and thus, the PHA produced by Synechococcus subsalsus has a larger number of potential industrial applications than that produced by

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Spirulina sp. LEB-18, especially in the packaging sector. 3.2.4 PHAs composition

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Table 7 presents the monomeric composition of PHAs produced by the two microalgae

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studied. A predominance of long chain building blocks, with 10 to 18 carbon atoms, was observed. The PHAs produced by the microalgae Synechococcus subsalsus

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presented a higher percentage of monomers with 16 (51.78%) and 18 (34.10%) carbon atoms, whereas the polymer produced by Spirulina sp. LEB-18 presented higher

AC

monomer content with 14 (16.49%) and 16 (72.58%) carbon atoms, demonstrating that the PHAs produced by each microalga present different monomeric compositions. A higher percentage of impurities (10.93%) was observed in PHAs extracted from Spirulina sp. LEB-18, confirming the results obtained in the thermogravimetric analyzes that indicated a higher PHA purity in the polymers produced by the cyanobacterium Synechococcus subsalsus. The detection of these building blocks is a genuine scientific novelty, given that absolutely novel PHA constituents are produced

25

ACCEPTED MANUSCRIPT by these microalgae. Although bacteria are capable of accumulating a greater amountquantity of PHAs, the polymers obtained are formed, in higher percentage, by monomers with 4 to 10 carbon atoms [7,10], presenting]. These higher monomer percentages present properties that hinder their industrial application.

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Table 7. Composition (% area/mass) of PHA determined by the GC-MS of

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Synechococcus subsalsus and Spirulina sp. LEB-18. Retention time

PHAs of Synechococcus

PHAs of Spirulina sp.

subsalsus

LEB-18

1.59

-

Identification NIST

SC

(min) methyl hydroxydodecanoate

5.84

methyl hydroxytetradecanoate

5.04

16.49

7.06

methyl hydroxyhexadecanoate

51.78

72.58

8.72

methyl hydroxyoctadecanoate

34.10

-

Other peaks

Unidentified

7.48

10.93

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D

MA

NU

4.72

The monomeric composition of PHAs is extremely dependent on the culture conditions and the producer strain, and is directly related to the properties of the polymers and their

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possible industrial applications. During the polymerization process, the strains showed

AC

the potential for improved adaptability to building blocks with 14 or 16 units of carbon. This shows the existence of a relationship between the molecular structure of the PHAs and the fatty acid composition of the biomass, where a higher content of long chain compounds (14-18 carbon atoms) was also observed, indicating that a common fatty acid metabolism intermediate serves as a precursor in the synthesis of PHA monomers. In this study, we have shown that the crystallinity of the polymeric material is very similar to that of the crystalline material [10,40], which was observed mainly in PHAs produced by Synechococcus subsalsus. The PHA produced by Spirulina sp. LEB-18 26

ACCEPTED MANUSCRIPT presented a lower percentage of long chain monomers, resulting in a polymer with higher crystallinity and lower molecular weight. According to Ribeiro et al. [10], the modulus and tensile strength of these copolymers decreases with increasing monomer concentration, and the elongation to break increases satisfactorily, demonstrating that PHAs obtained have great potential for industrial application. These data demonstrate

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that the PHAs obtained have great potential for industrial application.

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4. Conclusion

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This study shows that nitrogen deficiency in the culture media for the microalgae Chlorella minutissima, Synechococcus subsalsus, and Spirulina sp. LEB-18 influences

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the metabolism of these microorganisms in order to reduce the production of primary metabolism compounds such as chlorophylls and proteins. Additionally, these bacteria

MA

divert their metabolic pathways to produce secondary metabolism bioproducts, such as lipids and polyhydroxyalkanoates. Although the microalgae Chlorella minutissima did

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not produce PHAs, even under nitrogen limitation, it became evident that the stress

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caused by nitrogen deficiency stimulates the production of biopolymers by the cyanobacteria.

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Polymers produced by Synechococcus subsalsus and Spirulina sp. LEB-18 presented different characteristics, evidencing the influence of the producing strain on PHA

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properties. The PHAs obtained are formed from long chain building blocks containing 14 to 18 carbon atoms. This monomeric composition is a scientific novelty, since it was not found in PHAs obtained from bacteria. The properties of the PHAs produced by the studied cyanobacteria stimulate the industrial application of these polymers, especially in the packaging area. 5. Acknowledgements

27

ACCEPTED MANUSCRIPT The authors acknowledge CNPq (National Council of Technological and Scientific Development) for financing the productivity project n ° 311392 / 2016-4, FAPESB (Foundation for Research Support of the Country of Bahia) by granting a doctoral scholarship and MCTI (Ministry of Science Technology and Innovation) for the

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financial support (n. 01200.005005/2014-49). 6. References

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ACCEPTED MANUSCRIPT Figure captions

Figure 1. Biomass concentration during Chlorella minutissima (A), Synechococcus subsalsus (B), and Spirulina sp. LEB-18 (C) cultures in standard and limited medium.

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Figure 2. Chlorophyll concentration during Chlorella minutissima (A), Synechococcus

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subsalsus (B), and Spirulina sp. LEB-18 (C) culture in standard and limited medium.

Figure 3. Production of polyhydroxyalkanoates during cultivation of Synechococcus

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Figure 4. Derivative thermogravimetrics (DTG) of the cultures biomass in standard and

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Figure 5. FTIR spectra of PHAs produced by the cyanobacteria Synechococcus

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Figure 6. Derivative thermogravimetrics (DTG) (A) and differential scanning

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calorimeter (DSC) analysis (B) of PHAs produced by cyanobacteria Synechococcus subsalsus and Spirulina sp. LEB-18.

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The influence of nitrogen deficiency on microalgae cultivation was evaluated.

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Microalgae growth was affected by nitrogen reduction in the crops.

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The PHAs synthesized presented novel properties and composition.

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