Exploitation of Nannochloropsis gaditana biomass for biodiesel and pellet production

Accepted Manuscript Exploitation of Nannochloropsis gaditana biomass for biodiesel and pellet production A. Cancela, L. Pérez, A. Febrero, A. Sánchez, J.L. Salgueiro, L. Ortiz PII:

S0960-1481(18)31259-X

DOI:

10.1016/j.renene.2018.10.075

Reference:

RENE 10725

To appear in:

Renewable Energy

Received Date: 20 December 2017 Revised Date:

24 April 2018

Accepted Date: 16 October 2018

Please cite this article as: Cancela A, Pérez L, Febrero A, Sánchez A, Salgueiro JL, Ortiz L, Exploitation of Nannochloropsis gaditana biomass for biodiesel and pellet production, Renewable Energy (2018), doi: https://doi.org/10.1016/j.renene.2018.10.075. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Exploitation of Nannochloropsis gaditana biomass for biodiesel and pellet production A. Cancelaa, L. Péreza,*, A. Febreroa, A. Sáncheza, J. L. Salgueiroa , L. Ortizb

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a

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Marcosende s/n, 36310, Vigo-Pontevedra, Spain. Tel.: +34-986-818685. e-mail: [email protected]

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(A. Cancela), [email protected] (L. Pérez*), [email protected] (A. Sánchez),

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[email protected] (J. L. Salgueiro).

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b

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Vigo, Campus A Xunqueira s/n., 36005 Pontevedra, Spain. [email protected] (L.Ortiz)

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*Corresponding author: L. Pérez

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Chemical Engineering Department, E.E Industrial, University of Vigo, Campus Lagoas-

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Department of Natural Resources and Environment Engineering, E E. Forestal, University of

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Harvested biomass

Transesterification

Oil content Biodiesel conversion

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ALGAL CULTURE

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Oil extraction

Fatty acids profile

Algal waste

Pellet

ACCEPTED MANUSCRIPT Abstract

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A comparison of different inorganic and organic flocculants at doses of 50, 100 and 200 mg/L

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was performed to achieve the maximum biomass harvesting of Nannochloropsis gaditana

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microalgae. The best results were reached by aluminium chloride with 90.9±0.2% biomass

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recovered followed by copper sulphate with 70.8±0.3% at the maximum doses used. If the

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behaviour is analysed after spending 24 hours, all flocculants recovered more than 90% algal

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biomass. In the oil extraction process, a maximum of 29.25±1.10% extracted oil was achieved

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by Soxhlet using methanol-chloroform 2:1 as solvent and applying previous disruption by

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microwave. 20.4% less oil was released when n-hexane was used. Ultrasound extraction

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assisted by microwave reached peak values of 22.60±1.03%. Methyl esters of saturated fatty

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acids (C14:0, C16:0 and C18:0) were found as the major constituents, accounting for about 70%

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of the total content. Direct transesterification with previous incubation accomplished higher

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biodiesel conversions than without it. Finally, pellet manufacturing from algal wastes obtained

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after the transesterification reaction was studied. The results indicated that these pellet should be

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mixed with another type of biomass (e.g., miscanthus or eucalyptus) to be used as fuel in

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biomass boilers.

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Keywords: renewable; energy; pellet; microalgae, harvesting, biodiesel

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Consumption and production of all types of fuel, increased up to historical records in 2014 [1].

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Oil remains the leading fuel in the world, 32.6% of global energy, increasing by 0.8 million

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barrels per day. An estimated global production of primary energy will increase to near 105.7

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quadrillion BTU in 2040 [2]. This means higher greenhouse gasses emission.

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On the other hand, the European Union has established an objective, known as horizon 2020,

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which supposes a decrease of 20% in CO2 emissions, a reduction of 20% in energy consumption

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and 20% of increment on renewable energies consumption. For these reasons, it is necessary to

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replace fossil fuels with alternative energy sources such as biofuels [3]. Biofuels are postulated

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as a short-term option and its production will grow steadily next years. Furthermore, biofuels

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could offer new opportunities to diversify income sources and fuel supply, to promote

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employment in rural areas and to increasing the security of energy supply [4].

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Biodiesel is an alternative diesel fuel, it is made from renewable biological sources such as

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vegetable oils and animal fats. It is biodegradable and nontoxic. It has low emission profiles and

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is environmentally friendly [5]. So, it is registered as an additive in fuel by Protection Agency

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Environmental (Enviroment Protection Agency - EPA - USA) and the European Union.

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Microalgae are a source of raw material for biodiesel production [6,7]. Microalgae contain a

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high percentage of protein (50% growth phase), amino acids, low cellulose and high lipids

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content. Lipids are divided into storage lipids and membrane lipids which can represent 50% by

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weight and they are soluble in many solvents. The TAG (triacylglycerol or neutral fat) are the

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most abundant of them. The rest are PUFAs (polyunsaturated fatty acids). Algae grow easily in

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almost all environments [4], presenting a much higher growth rate and productivity than

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conventional forestry, agricultural crops and other aquatic plants [8]. Nannochloropsis gaditana

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is one of the most widely used microalgae species for biodiesel production. High rates of

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biomass productivity and lipid content are shown by this microalgae genus (300 mgL-1day-1 and

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45%, respectively) according to [9]. Authors such as [10] have obtained conversion of fatty

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acids to methyl esters of more than 90%.

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It is a marine alga that was first isolated in Cadiz (Lubián, 1982) [11]. It is difficult to identify

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because of its small size. In growth phase, cells are ellipsoidal shaped of 3.5-4 x 2.5-3 µm. They

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are immobile, don’t have flagella but have a parietal simple green chromatophores, occupying

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the greatest area of the cell. The cytoplasm has high lipid accumulation. The cell wall is smooth,

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thick and strong. Nannochloropsis is a major source of chlorophyll, zeaxanthin, canthaxanthin

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and astaxanthin production as well as polyunsaturated fatty acids (PUFAs) of interest

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recognized [12]. The disruption method applied to the Nannochloropsis species must be

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Introduction

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ACCEPTED MANUSCRIPT carefully chosen since the thick cell wall makes difficult the intracellular extraction [13].

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Different cell disruption methods can be used (e.g. ultrasound, microwave or supercritical

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fluids) depending on the scale at which the oil extraction or biodiesel obtaining is carried out

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(commercial scale or laboratory scale).

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Methods such as Soxhlet extraction and ultrasound are widely used on a small scale. However,

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numerous limitations are shown (several purification steps, generation of large amounts of

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waste or longer reaction time). These limitations hinder the use of these lipid extraction

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techniques or biodiesel production on a commercial scale. Numerous efforts have been made to

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achieve highly efficient and economically viable alternatives. Among them, supercritical fluid

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extraction method (e.g., supercritical carbon dioxide) stands out for its extraordinary efficiency

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of lipid extraction and for obtaining high value pigments that make it a competitive economic

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process [14,15]. On the other hand, hydrothermal liquefaction (HTL) is considered a promising

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technology for the production of renewable fuels on a large scale. The water is used as reaction

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medium, which allows wet feedstock to be used, eliminating drying costs [16]. In addition, this

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process could be optimized by catalytic hydrothermal gasification (CHG) and recycling of

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nutrients [17]. CHG can be used to clean up the organic material present in the resulting

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aqueous product to produce methane rich or hydrogen rich syngas.

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In this paper the viability of energy uses of Nanochloropsys gaditana, for simultaneous liquid

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and solid fuels (biodiesel and pellets) production is analysed. Aspects as separation of

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microalgae from culture medium, oil extraction, biodiesel synthesis by transesterification and

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finally pellets manufacturing from the remaining biowaste are studied at laboratory scale.

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Materials and methods

2.1 Microalgae cultivation

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Algae grew in polyethylene 45 L capacity photobioreactors in the Marine Science Station of

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Toralla (ECIMAT) using natural filtered seawater. GoldMedium (Aqualgae) was used as culture

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medium. The cultivation time was 15 days at 21±1 ºC under 17/7 light/dark cycles, the average

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size was 4 µm, the pH 8.11 and the cell accounting at the end of culture was 21000 cell/µl.

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2.2 Harvesting

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Harvesting process is a key step in obtaining microalgae biomass because it can lead to a 20 to

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30% of the total production cost due to the high energy required [18–20]. The low cellular mass

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density (typically in the range 0.3 to 5 g/L) and small algal size (between 2 -40 µm) make

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ACCEPTED MANUSCRIPT difficult this operation. The separation of Nanochloropsys gaditana microalgae from its culture

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medium has been conducted in this research. Three different coagulants were used in order to

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determine the best for an effective algae cells coagulation and subsequent sedimentation. The

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coagulants used were copper sulphate pentahydrate (CuSO4·5H2O), agar-agar and aluminium

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trichloride hexahydrate (AlCl3·6H2O) in concentrations of 50 mg/L, 100 mg/L and 200 mg/L.

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The procedure began with the addition of coagulant to the medium, followed by one minute

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stirring at 200 rpm and then three minutes at 50 rpm on HANNA HI 190 M Magnetic Mini-

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Stirrer. During the first minute the coagulant was mixed throughout the culture volume and

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subsequently algae junctions were formed with the coagulant. All flocculation experiments were

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carried out in 1L glass beakers. Absorbance measurements of the samples were taken from the

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top of the container throughout 200 minutes and at 24 hours after adding flocculants. The

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absorbance of the samples was determined at the wavelength of 680 nm by Labomed Spectro 22

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spectrophotometer.

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With the aim of determining the flocculation efficiency of the different coagulants used, these

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experiments were compared with a blank (without the addition of flocculants). In addition, two

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different samples were used as blank, one at 21 ºC and another at 4 ºC in order to determine the

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effect of the temperature on the sedimentation rate. Flocculation efficiency was calculated

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according to Eq. 1:

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% Biomass Recovery = 1 − / x 100

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Where, A=Absorbance value of sample and B= Initial absorbance value. 2.3 Dewatering

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Once the algal biomass has been harvested, it was dried in a Selecta Conterm 2000208 oven to

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reach low water content, around 20%. Then, the algal biomass was washed with distilled water

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to remove salts or traces of the flocculants used. Finally, algae were separated from the aqueous

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medium by centrifugation using Selecta Mixtasel centrifuge. The samples were centrifuged at

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1485 g (4000 rpm) for 15 minutes twice.

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2.4 Oil Extraction

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Oil extraction experiments were performed in order to characterize the algal oil. Two methods

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for lipid extraction, Soxhlet and Ultrasound (US), were employed and their performance were

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quantified. For that, wet microalgal biomass (79.31% moisture) was used. Some of the

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extraction experiments were preceded by cell disruption using microwaves (MW) [21]. One

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ACCEPTED MANUSCRIPT way to perform cell disruption was insert a round-bottom flask with wet algal biomass in a

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modified Becken Easycook Digital 2 microwave oven for 1 min (at 10 second intervals) and

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700 W power. In the other route followed, the biomass was maintained for 15 min (at 1 min

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intervals) and at 140 W (20% power) in the MW. The microwave reactor was equipped with a

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reflux condenser with the aim of prevent liquid splashes. Two different solvents were used to

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compare their performance, n-hexane and methanol-chloroform 2:1.

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In soxhlet extraction, the biomass was loaded in a Soxhlet apparatus and subjected to a series of

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cycles for 4 hours. On the other hand, ultrasound extractions took place in an ultrasonic unit

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Elmasonic S300H set to 37 kHz for 60 minutes. The samples were loaded into 500 mL three-

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neck round-bottom flasks. During the sonication experiments, the variation of the temperature

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inside balls and inside the bath was measured. Vacuum filtration was used to separate the solid

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matter from the extracted product. Then, the liquid mixture of oil-solvent obtained by both

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methods, was evaporated in an oven at 105 °C. Oil samples were characterized by gas

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chromatography. The wet weight of the samples used in all experiments conducted, varied

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between 8.5 and 20 g. The characteristics of the different tests carried out can be observed in

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Table 1.

Soxhlet

Test name S1

Extraction time (hours)

S2

4

S3

132 133 134

US2

Ratio solvent/algae

n-hexane

150:1 (v/dw)

n-hexane

150:1 (v/dw)

15 min, 140W methanol-chloroform 2:1 35:1 (v methanol/dw)

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NO

methanol-chloroform 2:1

5:1 (v methanol/dw)

1 min, 700W

methanol-chloroform 2:1

5:1 (v methanol/dw)

Table 1: Characteristics of the different tests carried out

Finally, oil extraction percentage was calculated according to Eq. 2:

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US1

1 min, 700W 15 min, 140W

Solvent

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US

MW Disruption

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Extraction method

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% Oil extraction =  − 0 x 100/

(2)

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Where, W=Weight of extraction vessel with oil expressed in g, W0=Weight of empty extraction

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vessel in g and DBW= Weight of dry algal biomass sample in g.

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2.5 Transesterification

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A direct production of biodiesel from Nannochloropsis gaditana microalga was carried out.

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Two types of catalysed in situ transesterification experiences were conducted (with and without

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a pre-incubation of the algae in methanol). The catalyst used was NaOH (sodium hydroxide)

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ACCEPTED MANUSCRIPT with a concentration of 2% by weight in methanol. Incubation was performed in 250 mL

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Erlenmeyer flasks for 18 hours at 21 ºC with half the amount of methanol. In both cases (with or

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without incubation), transesterification reaction was carried out at constant temperature (60 °C)

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and atmospheric pressure in an OVAN OPAQ I10-0E incubator and OVAN MAXI OL30-ME

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orbital shaker. The transesterification reaction was conducted for 240 minutes under constant

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stirring (160 rpm). During the reaction 5.25 g wet algal sample (79.31% moisture) was allowed

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to react with 58:1 ratio methoxide/algae (v/dw). It must be taken into account that the

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stoichiometric ratio alcohol:triglycerides is 3:1, however higher alcohol ratios are required in

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order to maximize the yield of the reaction and shift the equilibrium towards the production of

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methyl ester (desired product) [22]. The amount of methanol used in the experiments was the

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minimum necessary to cover the algal sample used. The recovered unreacted methoxide from in

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situ transesterification could be recycled by neutralization and subsequent methanol evaporation

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[23]. The recovered methanol could be used again as a solvent.

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Finally, the biodiesel was decanted and separated from glycerol. Biodiesel density was

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calculated to find out if it fulfils UNE-EN 14214 standard [24].

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2.6 Pelletization and calorimetric determination

Transesterification process wastes were characterized in terms of use as pellets [25]. The

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properties analysed were humidity content, volatile matter, ash and fixed carbon according to

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UNE-EN 14774-2 [26], UNE-EN 15148 [27], UNE-EN 14775 [28] respectively, while fixed

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carbon was calculated by difference. A Parr 1261 bomb calorimeter was also used to calculate

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the heating value following UNE-EN 149187 [29]. The analyses were performed in triplicate.

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Results and discussion

3.1 Harvesting

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Control test by gravity sedimentation at 21 ºC and low temperature (4 ºC) has been tested for

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Nannochloropsis gaditana microalgae without flocculants addition. The best results were found

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at 21 ºC (Figure 1).

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Figure 1: Gravity sedimentation of Nannochloropsis gaditana

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Microalgae recovery efficiency was 35% in the first seven hours at room temperature and 17%

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in the same time at low temperature. After 24 hours, a recovery efficiency of 80% was achieved

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at 21 ºC and 150 hours were necessary to approach recovery efficiency values of 95%. At that

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time (150 h), the sample of low temperature reached a recovery of 68% and needed 13 days to

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manage algal biomass recovery of almost 100%. These data showed that flocculants addition is

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necessary to ensure a harvesting process faster.

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Copper sulphate, agar-agar and aluminium trichloride were employed in three different doses,

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50 mg/L, 100 mg/L and 200 mg/L. The best results were accomplished by AlCl3.6H2O and

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CuSO4.5H2O respectively, at the highest concentrations. As can be seen in Figure 2(a), 50%

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biomass recovery was reached between 5 and 10 minutes when the trivalent metal ion of AlCl3

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was added. In the same way, in less than 20 minutes 80% recovery efficiency was managed.

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These data are in concordance with those showed by Surendhiran and Vijay [30], who obtained

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85.46% biomass recovery when Nannochloropsis Oculata was flocculated with AlCl3. For its

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part, Garzon-Sanabria et al. [31] reached a higher than 90% removal efficacy at aluminium

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trichloride dose of 50 mg/L after 5 min for the same microalgae species.

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80% 60% 40% 20%

80% 60% 40% 20%

0% 0

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Biomass recovery

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100%

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100%

Biomass recovery

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100

time (min)

200

0%

AlCl3 50 [mg/L]

AlCl3 100 [mg/L]

AlCl3 200 [mg/L]

CuSO4 50 [mg/L]

CuSO4 100 [mg/L]

CuSO4 200 [mg/L]

Agar-agar 50 [mg/L]

Agar-agar 100 [mg/L]

Agar-agar 200 [mg/L]

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Figure 2: Biomass recovery of Nannochloropsis gaditana (a) during 230 min, (b) at 24 h

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ACCEPTED MANUSCRIPT 192 However, the organic flocculant agar-agar seemed to be inefficient to flocculate

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Nannochloropsis gaditana microalgae during the first 4 hours (Figure 2(a)). Recoveries after 24

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hours were shown in Figure 2(b). Contrary to expectations, recoveries above 90% were reached

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by this natural flocculant at doses of 100 and 200 mg/L. Very similar recoveries were shown by

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Sadegh et al. [32] when another organic flocculant, chitosan (100 mg/L), was used with

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Nannochloropsis sp. Therefore, efficient and rapid harvesting was found when aluminium and

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copper salts were used, nevertheless larger times were required by agar-agar.

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3.2 Oil Extraction and characterization

In the current research, two different extraction methods, Soxhlet and ultrasound, have been

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conducted. The results obtained are showed in Figure 3. Oil contents between 3.7% and 29.3%

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have been recovered from Nannochloropsis gaditana microalgae through 4 h of Soxhlet

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extraction and pre-disruption by microwave. It can be observed that cell disruption is more

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effective with longer times and lower power. Thus, when n-hexane was used as solvent, 5%

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more oil was extracted for tests at a 140W power and 15 minutes than tests carried out at 700 W

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power and 1 minute. However, this non-polar solvent has shown extraction yields much lower

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than those achieved by 2:1 methanol-chloroform mixture (29.25± 1.10%). Thanks to the

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combination of a polar and a non-polar solvents, more oil is released. Thus, polar lipids are

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extracted by methanol while neutral lipids are extracted by chloroform. Based on data published

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by other authors, the percentage of extracted oil can reach up to 49% when methanol is replaced

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by ethanol in this binary mixture for Nannochloropsis sp.[33].

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met-chlor. 2:1 met-chlor. 2:1

20 15 10 5

n-hexane met-chlor. 2:1

n-hexane

0

213 214

Soxhlet (MW 700W, 1 min)

Soxhlet (MW Soxhlet (MW 140W, 15 min) 140W, 15 min)

US (No MW) US (MW 700W, 1 min)

Figure 3: Oil values of Soxhlet and ultrasound extractions

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On the other hand, two types of experiments were performed using ultrasound extraction. The

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mean values accomplished from the different ultrasound extractions showed that previous

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ACCEPTED MANUSCRIPT microwave disruption improved recovery, a total 22.60± 1.03% extracted oil of total dry

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biomass was reached, while the experiences with no previous disruption obtained values of

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6.22± 0.15%. The temperature variation of sonication bath and the reaction temperature

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variation for the experiences with disruption and without disruption are shown in Figure 4. The

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values of temperature variation with time were correlated by linear fit. Studies performed by

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other authors have demonstrated better lipid yields by using microwave cell disruption [34].

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Data reported by Lee et al. [35], suggest that the lipid extraction yield is highly influenced by

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the microalgal species and the type of pre-treatment applied. Finally, based on the results

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obtained in this research, it can be concluded that although US extraction method showed

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similar results to the Soxhlet method (both with previous MW disruption) lower ratio solvent-

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algae and less time were required by ultrasound.

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40 Temperature (ºC)

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35

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20 0

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20

30 40 time (min)

Batch Temperature (NO MW) Reaction Temperature (NO MW) Bath Temperature (MW) Reaction Temperature (MW) 50

60

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Figure 4: Temperature variation in oil extractions with and without MW disruption

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After lipid extraction, the fatty acids composition of Nannochloropsis gaditana biomass was

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determined by gas chromatography. Methyl esters of saturated fatty acids were found as the

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major constituents, accounting for about 70% of the total content. This composition indicates

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that the biodiesel obtained from Nannochloropsis gaditana microalgae could have a good

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resistance to oxidation and stored for long periods of time [36]. Methyl esters of palmitic acid

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were revealed as the dominant fatty acid in the studied algal species (45.80±0.38%), followed

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by palmitoleic (18.30±0.53%) and stearic acid (17.07±0.31%). Small amounts of myristic, oleic,

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linoleic and ecosapetaenoic acids were also detected (6.83± 0.06, 4.48 ± 0.04, 1.19 ± 0.12 and

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6.33± 0.37, respectively). The results obtained in this research are in agreement with those

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shown by Perrier et al. for Nannochloropsis microalgae [37]. Lower contents of C16:0 and

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C18:0 were achieved, however higher percentages of unsaturated fatty acids such as C16:1,

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C18:1 and C20:5 were reached in their study. Nevertheless, when another similar marine

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species, Nannochloropsis Oculata, was analyzed by Sung-Suk et al. [38], variation in the

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saturated fatty acids content was appreciated. Stearic acid was present in a higher amount while

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lower proportion of myristic and palmitic acid were detected. It can be concluded that the fatty

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acid profile obtained in the present research, is typical of green algae [33]. 3.3 Biodiesel and Pellet production

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Direct transesterification yields, with and without incubation, were analysed. When direct

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transesterification with methanol and sodium hydroxide was conducted for 4 hours at 60 °C,

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77±1% conversions of triglycerides to biodiesel were achieved. However, when 18 hours of

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incubation were previously applied to the transesterification reaction, higher conversions were

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reached (80±2%). Similar values were obtained by Kober et al. when Nannochloropsis

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microalgae was subjected to regular reflux technique without initial extraction step [36].

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Microalgal biodiesel must meet standard specifications for its use in combustion engines. In the

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United States, it is the ASTM D 6751-6 standard that sets these requirements while the

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European Union is governed for the UNE-EN 14214 standard [24]. Density of the synthetized

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biodiesel was calculated. It was found that a value of 0.89±0.01 g/mL was managed when the

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transesterification reaction was subjected to previous incubation. This value accomplishes the

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European and American Standard. However, lower density values (0.83±0.02 g/mL) were

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obtained without incubation, not meeting the requisites established. Density is an important

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biodiesel property because it influences the atomization efficiency of the combustion system

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and it could cause poor combustion in chamber [39]. The results suggested that other

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physicochemical properties such as viscosity, cetane number or flash point should be checked in

263

order to know if the biodiesel from Nannochloropsis microalgae is valid for its use in

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combustion engines.

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By other hand, the possibility to use transesterification process wastes for making pellets was

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studied. Pellet properties were analysed with the aim of verifying the European standard for

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their use as fuel in biomass boilers [40]. These standards are referred to wood pellet. The data

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managed for algae pellets are shown in Table 2. The requirements set by the regulations are:

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humidity content (wt%w.b) ≤10%, ash content (wt% d.b) ≤0.7 and low heating value (MJ/kg

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≥16.5). Based on the results obtained, only the humidity content was verified. An ash content

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much higher than that indicated in the regulations was found in algal pellets while the calorific

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value was slightly lower than the required value. The high ash content may be due to the fact

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that Nannochloropsis gaditana microalgae has been harvested by flocculation and cations such

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as Cu2+ and Al3+ were present in the analysed biomass [41]. These data are in agreement with

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those obtained by Cancela et al. when other algal species such as Scenedesmus sp.,

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Kirchneriella sp., and M. Aeruginosa were used to make pellets [42]. Therefore, the pellets

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obtained could not be used directly in biomass boilers. However algal biomass could be mixed

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with other biomass sources such as Miscanthus or Eucalyptus, achieving the values established

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in the regulations. (wt% w.b)

0

Fixed Carbon

(wt% d.b)

9.718±0.005

Volatile matter

(wt% d.b)

58.822±0.003

Ash

(wt% d.b)

30.460±0.004

HHV

(kJ/kg)

17320.923

LHV

(kJ/kg)

15998.461

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Table 2. Analysis of algal waste pellet

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Inorganic flocculants such as aluminium chloride or copper sulphate have shown higher algal

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biomass recovery efficiencies than the organic flocculant agar-agar for short periods of time.

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Regarding the results of biodiesel and oil production achieved in this research, they are not in

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line with the desired characteristics. However, future studies to produce biodiesel or extract

287

lipids from Nannochloropsis gaditana microalgae could be improved by employing more

288

efficient techniques and more adequate. On the other hand, the analysis of the properties of

289

pellets manufacturing from the residue resulting of transesterification reaction have shown that

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they should be mixed with another type of biomass to be used as fuel in biomass boilers.

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Another option for future research is the exploitation of carbohydrate for the production of

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monomeric carbohydrates.

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Conclusion

Acknowledgement

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The authors would like to thank ECIMAT (Estación de Ciencias Marinas de Toralla) belonging

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University of Vigo, Spain, for providing the cultures of Nannochloropsis gaditana microalgae.

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ACCEPTED MANUSCRIPT Aluminium chloride is highly efficient for Nannochloropsis Gaditana flocculation

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Microwave disruption improves Soxhlet and ultrasound extraction yield

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Methyl esters of saturated fatty acids are the major oil constituents

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Direct transesterification with incubation reached the highest biodiesel conversion

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Algal waste pellet could not be used directly used as fuel in biomass boilers

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