Screening of marine microalgae isolated from the hypersaline Bardawil lagoon for biodiesel feedstock

Accepted Manuscript Screening of marine microalgae isolated from the hypersaline Bardawil lagoon for biodiesel feedstock

Abd El-Fatah Abomohra, Mostafa El-Sheekh, Dieter Hanelt PII:

S0960-1481(16)30879-5

DOI:

10.1016/j.renene.2016.10.015

Reference:

RENE 8203

To appear in:

Renewable Energy

Received Date:

19 January 2016

Revised Date:

01 July 2016

Accepted Date:

06 October 2016

Please cite this article as: Abd El-Fatah Abomohra, Mostafa El-Sheekh, Dieter Hanelt, Screening of marine microalgae isolated from the hypersaline Bardawil lagoon for biodiesel feedstock, Renewable Energy (2016), doi: 10.1016/j.renene.2016.10.015

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1. Isolation of halophilic microalgae from the hypersaline Bardawil lagoon for biodiesel production. 2. The green microalga Tetraselmis elliptica showed the highest lipid and fatty

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acid productivities. 3. Fatty acid profile and iodine value of Tetraselmis elliptica within the European standard specifications.

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4. Tetraselmis elliptica is a promising species as biodiesel feedstock.

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Screening of marine microalgae isolated from the hypersalineBardawil lagoon for

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biodiesel feedstock

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Abd El-Fatah Abomohraab, Mostafa El-Sheekha*, Dieter Haneltb

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aPhycology

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31527 Tanta, Egypt

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bDepartment

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D-22609 Hamburg, Germany

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Research Unit, Botany Department, Faculty of Science, Tanta University,

of Cell Biology and Phycology, University of Hamburg, Ohnhorststrasse 18,

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*Correspondence :[email protected]

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Abstract

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Recently, microalgae have been attracting a wide attention as a source of high-lipid

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feedstock to produce biodiesel. A total of twenty one halophilic microalgae were isolated

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from the hypersalineBardawil lagoon North Sinai, Egypt. Nine of them were further

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characterized with respect to biomass and fatty acid productivities. Biomass productivity

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as cellular dry weight (CDW), fatty acid content and, consequently, fatty acid

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productivity of the chlorophyteTetraselmisellipticawas the highest among alltested strains

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(0.122 g CDW L-1 d-1, 77.36 mg g−1 CDW and 14.1 mg L-1 d-1, respectively). Lipid

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fractionation showed that total lipids represented 12.96 mg g-1 CDW and neutral lipids

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represented 37 % of the total lipids with corresponding iodine value of 70.3 g I2/100 g oil.

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In all fractions, C16:0 and C18:1n-9 were predominant, being as high as 31 and 20 % of

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total fatty acids in neutral lipids, 26 and 24 % of total fatty acids in polar lipids and 28

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and 26 % of total fatty acids in phospholipids, respectively. This study demonstrates that

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the halophilic microalga T. ellipticaisolated from hypersaline water is a promising species

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for biodiesel feedstock.

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Keywords: Microalgae, Screening, Neutral lipid, Tetraselmiselliptica, Biodiesel

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1. Introduction In order to realize a stable energy alternative to the present sources that will meet

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world demand while mitigating climate change through CO2 sequestration and emissions

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reduction, it is required to extendclean renewable fuels. Ironically, most renewable

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energy initiatives were focused on electricity generation, while about two thirds of world

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energy consumption is derived from liquid fuels [1]. Therefore, biomass-derived liquid

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fuels are receiving greater attention; and with predictions that crude oil prices will reach

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record

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attention[2,3,4].Microalgae are unicellular or multicellularphotosynthetic microorganisms

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that convert sunlight, water and carbon dioxide into biomass. They can be found in

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diverse of environments and harsh conditions, living in saline or freshwater environments

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[5]. Microalgae can provide several different types of renewable biofuels, including

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anaerobic digestion of the algal biomass into methane, oil transesterificationinto

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biodiesel, saccharification of carbohydrates into ethanol, cracking of hydrocarbons and

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isoprenoidsinto gasoline and direct photobiologically synthesis of biohydrogen[6-11]. In

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recent years, usage of microalgae as biodiesel feedstock has attracted great attention [12-

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14]. Using of microalgae as a biofuel feedstock was first proposed in 1950s; and since

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1970s several funded research programs in different countries were started to study the

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efficiency of microalgae in biodiesel production [15].Microalgae have been cited as one

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of the best non-edible feedstock for biodiesel compared to oleaginous crops, such as

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soybean, rapeseed and oil palm. The priority of microalgae in biodiesel production is due

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to 1) higher oil productivity which at least 15-20 times higher than conventional crops, 2)

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high biomass production due to short doubling time, 3) does not necessarily require

highs,

algal

based

biofuels

are

gaining

widespread

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breaking

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arable land for growth, 4) high CO2 sequestration rate and wastewater treatment and 5)

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require much less land areas compared to conventional crops [16-17]. In addition to the

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previous advantages, marine microalgae have additional preference that they don’t

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compete for freshwater.

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Bardawil Lagoon is one of the five northern lakes in Egypt. It is bordered from the

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south by a sand dune belt and from the north by a convex sand barrier that separates it

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from the Sinai Mediterranean coast (Figure 1). The area of that lake is about 685 km2 and

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extends for a distance of 80 km with maximum width of 20 km and maximum depth of 3

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m [18]. The water temperature values fluctuated between a minimum value of 11.6 ºC

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during January to a maximum value of 33.2 ºC in July, with annual mean of 21.5 ± 6.5 ºC

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and has salinity much higher than that of the open sea [19]. The key technical challenge

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in biodiesel production by microalgae is to identify a strain with the highest growth rates

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and oil contents with suitable fatty acid profile. The present study examines the efficiency

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of marine microalgae isolated from Bardawillagoon as a source of biomass and biodiesel

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productionin terms of lipid content and fatty acids productivity.

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Fig. 1. Map of the northern side of Sinai Peninsula showing the location of the Bardawil

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Lagoon. Source: satellite picture taken with Google earth.

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2. Materials and methods

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2.1. Sampling site

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Figure2 shows a schematic diagram of the isolation and screening procedures of

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microalgal strains in the present study.Microalgae were isolated from three water samples

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collected during April 2013 from Marsa El-Nasr (N 31º 05\ 26\\, E 32º 52\ 17\\),

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Bardawillagoon, Sinai Peninsula, Egypt. The samples were collected in plastic bottles

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and brought to the laboratory shortly after collection. Temperature, pH, water depth,

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turbidity, salinity and water transparency were measured in the field. Water transparency

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was measured using Secchi disc of 25 cm in diameter. Some water parameters were

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measured directly after collection (Na, K, Ca, Mg, total N, total P, Mg, K, Na and Ca).

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Atomic absorption spectrophotometer (Shimadzu AA-6300) was used for determination

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of P, Mg, K, Na and Ca. Molybdenum blue and indo-phenol blue methods were applied

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for the determination of total P and total N, respectively, using a spectrophotometer

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(CECIL CE 1021). All these procedures are outlined in APHA/AWWA/WPCF [20].

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Fig. 2.Schematic diagram for the procedures of isolation and screening of microalgae for

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biodiesel production.

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2.2. Isolation, purification and identification of microalgae

Isolation and purification of microalgae was performed on f/2 medium [21] by

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sub-culturing as previously described by Robert [22]. A total of 21 marine microalgal

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strains were isolated from the collected water samples in unialgal cultures, however nine

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strains were chosen after the first selection (Figure 2) depending on their relatively higher

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growth. Selected strains were identified using their morphological features and deposited

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in Tanta University Culture Collection, Tanta, Egypt.

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2.3. Growth and biomass assay

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For selection of media providing the best growth, each strain was individually

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cultivated in 100mL Erlenmeyer flasks at 20 ± 1 °C. For this purpose, six different

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marine media were used (Table 1). The Flory medium was prepared by dissolving of 2 g

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of N-free Flory Basic Fertilizer 1 (Euflor, Germany) and 810 mg potassium nitrate as

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nitrogen source in 1 L of natural seawater. Optical density (OD540) was measured after 12

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days of growth to identify the best medium for the growth of each microalgal strain.

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Table 1. Different media used for cultivation of the isolated species. Medium common name

Given abbreviation

Reference

Silicate enriched sea water

SES

EPSAG*

F/2

F/2

Modified Artificial Seawater

MAS

enriched natural seawater

ES

Flory Basic Fertilizer 1

FM

brackish water medium with

[21]

CCAP** [39]

provided by Euflor, Germany EPSAG*

*EPSAG refers to Culture Collection of Algae, Goettingen, Germany ** CCAP refers to Culture Collection of Algae and Protozoa, Oban, UK

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BMS

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A certain volume of exponentially growing microalga,precultured in 1 L

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Erlenmeyer flasks on the best medium, was inoculated in 300 mL of the corresponding

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medium in Kniese tubes [13] at an initial OD540 of 0.06. Sterile filtered air enriched with

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1.5 % (v/v) CO2 was continuously applied to all cultures. Algal growth was monitored

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using the optical density of the culture at 540 nm (OD540) and by determination of algal

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cellular dry weight (CDW). Biomass productivity was calculated according to Abomohra

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et al. [14].

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2.4. Lipid extraction

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Extraction of lipids was performed using chloroform:methanol (2:1)according to

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Folch et al. [23]. Lipid extracts were dried under a stream of argon. The pre-weighed

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glass vials containing the lipid extracts were dried at 80 °C for 30 min, cooled in a

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desiccator and weighed again.

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2.5. Fatty acid profiles To analyze the intracellular fatty acid composition, 5 ml aliquots of each culture

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were collected at times specified. Lipids were extracted following the method of Bligh

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and Dyer [24]. Prior to extraction, trinonadecanoylglycerolwas added to the samples as

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internal standards. To determine the fatty acid profiles, esterified fatty acids from the

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extracts of intracellular lipids were converted to fatty acid methyl esters

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(FAMEs)bytransmethylationas described previously [25-26]. FAMEs were subjected to

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analysis by GC (Varian 3900 GC-system equipped with a Varian capillary column, Select

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Fame, 50 m length and 0.25 mm internal diameter); and FAME productivity was

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calculated according to Abomohra et al. [14].

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2.6. Fractionation of lipids

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Lipid fractionation was performed to the total lipid extract of the promising

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microalga according to the method described by Fakas et al. [27]. Briefly, 50 mg of total

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lipid extract were dissolved in 1 ml chloroform, and fractionated using a column (25 mm

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× 100 mm) containing 1 g silicic acid activated by heating overnight at 80 °C. Successive

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applications of 100 ml dichloromethane, 100 ml acetone and 100 ml methanol produced

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fractions containing neutral lipids, polar lipids and phospholipids, respectively. Lipid

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fractions were collected respectively and carefully weighed after solvent removal.

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Individual fatty acid proportions were measured in each lipid fraction using GC as

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described in the previous chapter. Degree of unsaturation (iodine number) for each

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individual fraction was measured as previously described by Abomohra et al. [11].

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2.7. Statistical analysis Results are presented as mean ± standard deviation (SD) from three replicates.

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The statistical analyses were carried out using SAS (v 6.12). Data obtained were analyzed

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statistically to determine the degree of significance using one-way and two-way analysis

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of variance (ANOVA) at probability level P≤ 0.05.

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3. Results

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Three water samples were collected around the location known as Marsa El-Nasr

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at Bardawil lagoon. Water at the collection sites showed neutral pH value of 7.43 with

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average temperature of 19.9 °C and 39.4 ‰ salinity (Table 2). Macronutrients analysis

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indicated relatively high concentrations of sodium (11538 mg L-1) followed by

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magnesium (1264 mg L-1), while total nitrogen and total phosphorus represented 75.6 and

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0.068 mg L-1, respectively (Table 2).

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Table 2.Some physico-chemical characteristics of the seawater at the collection sites.

pH

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Characteristics

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7.43 ± 0.15 19.9 ± 0.4

Water depth (cm)

24.0 ± 3.0

Turbidity (cm)

24.0 ± 3.0

Salinity (‰)

39.4 ± 3.2

N (mg L-1)

75.6 ± 7.6

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Temperature (°C)

P (mg L-1)

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Concentration

0.068 ± 0.005

Mg (mg L-1)

1264 ± 46

K (mg L-1)

704 ± 18

Na (mg L-1)

11538 ± 865

Ca (mg L-1)

22.6 ± 2.3

Values are the mean of reading at three collection sites ± SD 9

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Out of total 21 isolated microalgae, 9 strains were selected on the basis of their

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relatively high growth which was noticed during isolation steps (first selection). In order

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to select a media that facilitates high biomass productivity in batch cultures, each strain

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was cultivated in six different media, and the growth was monitored by OD540 after 12

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days of incubation (Table 3). Chlorella vulgaris,andTetraselmisellipticashowed

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maximum growth on FM, while Cyclotella sp., Dunaliellasalinaand Navicula sp.showed

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maximum growth on SES. F/2 was recorded as the best medium for growth of

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Naviculamolli, Chlorella sp.1,Chlorellasp.2 and Pinnularia sp.(Table 3). Consequently,

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only the medium providing highest growth for each strain was used for further

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experiments.

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Table 3. Growth of isolated microalgae (as OD540) cultivated in different growth media

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for 12 days.

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Growth (OD540)

Microalgae SES

F/2

MAS

ES

FM

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BMS 0.219 ± 0.013a

0.121 ± 0.015b

0.113 ± 0.012b

0.187 ± 0.010a

0.233 ± 0.009a

0.281 ± 0.017c

T. elliptica

2.420 ± 0.090a

2.540 ± 0.082b

2.817 ± 0.099c

3.293 ± 0.064d

3.540 ± 0.070e

3.877 ± 0.121f

N. molli

0.095 ± 0.005a

0.284 ± 0.010b

0.381 ± 0.011c

0.130 ± 0.006a

0.101 ± 0.006a

0.219 ± 0.0d

Cyclotella sp.

0.088 ± 0.010a

0.296 ± 0.011b

0.256 ± 0.008bc

0.235 ± 0.007cd

0.141 ± 0.008a

0.186 ± 0.006d

Chlorella sp.1

0.057 ± 0.006a

0.252 ± 0.009b

0.288 ± 0.009b

0.137 ± 0.005c

0.102 ± 0.004ac

0.154 ± 0.009c

Chlorella sp.2

0.051 ± 0.007a

0.294 ± 0.008b

0.345 ± 0.009b

0.203 ± 0.010c

0.097 ± 0.008ad

0.134 ± 0.012d

D. salina

0.057 ± 0.004a

0.237 ± 0.009b

0.187 ± 0.008abc

0.078 ± 0.010d

0.113 ± 0.012de

0.147 ± 0.008ce

0.119 ± 0.015a

0.555 ± 0.023b

0.641 ± 0.015c

0.108 ± 0.014a

0.319 ± 0.012d

0.411 ± 0.019e

0.104 ± 0.012a

1.463 ± 0.046b

1.191 ± 0.034c

0.346 ± 0.026d

0.666 ± 0.028e

0.889 ± 0.038f

Navicula sp.

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Pinnularia sp.

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C. vulgaris

Each value is the mean of three replicates ± SD. Values with the same letter in the same row are not significant (at P≤ 0.05).

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The duration of exponential phase varied between 8 days for N. molli, Cyclotella

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sp.;and D. salinato 16 days for Navicula sp. (Table 4). T. elliptica showed highest

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biomass productivity of 0.122 g CDW L−1d−1 which was 31, 50, 52 and 54 % higher than

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Cyclotella sp.,Chlorella sp.1, Chlorella sp.2 andPinnularia sp., respectively. While C.

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vulgarisshowed the lowest biomass productivity of 0.016 g CDW L−1d−1 (87 % lower

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than T. elliptica, Table 4).

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Table 4. Biomass production of different isolated microalgae Cellular dry weight (g L-1)

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Biomass

Duration of

Growthmedia

At earlyexponential

productivity

Atlate exponential

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0.016 ± 0.008a

2.394 ± 0.068

12

0.122 ± 0.006b

0.707 ± 0.026

8

0.041 ± 0.003c

0.883 ± 0.022

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0.085 ± 0.003d

0.220 ± 0.057

0.827 ± 0.056

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0.061 ± 0.006e

F/2

0.386 ± 0.029

0.862 ± 0.030

8

0.059 ± 0.004e

D. salina

SES

0.119 ± 0.034

0.482 ± 0.072

8

0.045 ± 0.009c

Pinnularia sp.

F/2

0.386 ± 0.028

1.162 ± 0.030

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0.056 ± 0.002e

Navicula sp.

SES

0.388 ± 0.027

0.772 ± 0.041

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exponential phase (d)

phase 0.889 ± 0.053

T. elliptica

FM

0.929 ± 0.168

N. molli

F/2

0.378 ± 0.052

Cyclotella sp.

SES

0.206 ± 0.008

Chlorella sp.1

F/2

Chlorella sp.2

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0.024 ± 0.003a

Each value is the mean of three replicates ± SD. Values of biomass productivity with the same letter are not significant (at P≤ 0.05).

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1.086 ± 0.098

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FM

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C. vulgaris

(g CDW L-1 d-1)

phase

Figure 3 shows FAME content and FAME productivity of the nine studies

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species, which showed significant differences between studied species (one way

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ANOVA, P= 0.0001 for both). Overall, T. elliptica showed the highest significant (one

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way ANOVA, P≤ 0.05) FAME content with a value of 77.36 mg g−1 CDW. However, C. 11

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vulgaris showed the lowest FAME content between the studied species (20.94 mg g-1

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CDW). As a result of high FAME content and high biomass production of T. elliptica, it

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showed the highest FAME productivity (14.1 mg L-1 d-1) which was 75 and 74 % higher

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than Chlorella sp.1 and Cyclotella sp. , respectively (Figure 3).

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Fig. 3.FAME productivities (grey bars) and FAME content (black dots) of different

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isolated microalgae during the exponential phase. Error bars show the SD of three

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replicates. Same letters for the same series indicate insignificant difference (at P≤ 0.05).

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Interestingly, although Navicula sp. showed relatively high FAME content (61.52

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mg g-1 CDW), it showed the lowest FAME productivity (0.361 mg L-1 d-1) due to its low

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biomass production (0.024 g L-1 d-1).Therefore, T. elliptica (Figure 4) was selected

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(second selection) as a promising halophilicmicroalga for further studies.

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Fig. 4.Photos of the isolated microalga Tetraselmiselliptica under light microscope (A)

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and fluorescent microscope with excitation wavelength of 540 nm (B).

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Fatty acid profile of T. ellipticawas studied at three growth points representing

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early exponential phase, middle exponential phase and late exponential phase.

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Insignificant changes (one way ANOVA, P=0.8213) were recorded in total fatty acid

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content from early exponential phase to late exponential phase (Table 5). However

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transition from early to late exponential phaseresulted in significant increase in the

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proportion of saturated and monounsaturated fatty acids by 31 and 52 %, respectively,

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with consequent 41 % significant decrease in polyunsaturated fatty acids (Table 5). The

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dominant fatty acids were 16-carbon and 18-carbon fatty acids, which showed

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pronounced variation in their concentration at different measurement points.

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Table 5.Fatty acid profile (as mg g−1 CDW) of Tetraselmisellipticaat different growth

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phases. Fatty acids

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Middle exponential phase

Late exponential phase

C14:0

0.18 ± 0.02

0.26 ± 0.01

0.35 ± 0.01

C16:0

15.34 ± 0.55

23.16 ± 1.25

21.90 ± 0.86

C16:1n-9

0.35 ± 0.04

0.24 ± 0.16

0.27 ± 0.05

C16:1n-7

1.49 ± 0.17

0.62 ± 0.02

0.77 ± 0.05

C16:2

1.12 ± 0.03

0.73 ± 0.03

0.77 ± 0.03

C16:3

1.10 ± 0.05

0.63 ± 0.02

0.70 ± 0.03

C16:4n-3

10.88 ± 0.55

7.17 ± 0.26

5.36 ± 0.63

C18:0

0.23 ± 0.01

0.34 ± 0.04

0.42 ± 0.01

C18:1n-9

10.22 ± 0.19

15.42 ± 0.55

18.00 ± 0.92

C18:2n-6

3.41 ± 0.06

2.73 ± 0.09

2.94 ± 0.12

C18:3n-3

13.69 ± 0.28

9.69 ± 0.31

8.88 ± 0.42

C18:3n-6

0.20 ± 0.01

C18:4n-3

8.19 ± 0.21

C20:0

1.94 ± 0.23

C20:1n-9

1.37 ± 0.01

C22:0 C24:0

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0.28 ± 0.03

4.70 ± 0.14

4.00 ± 0.13

1.99 ± 0.08

2.46 ± 0.10

1.12 ± 0.10

1.44 ± 0.09

6.42 ± 0.09

5.10 ± 0.15

7.39 ± 0.50

1.82 ± 0.02

1.07 ± 0.03

1.45 ± 0.05

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0.25 ± 0.02

SFAs

25.92 ±

MUFAs

13.43 ± 0.51a

17.40 ± 0.98b

20.48 ± 1.61c

PUFAs

38.58 ± 1.18a

25.90 ± 0.86b

22.92 ± 1.38c

Total

77.94 ± 2.04a

75.23 ± 3.02a

77.37 ± 3.68a

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31.93 ±

1.41b

33.96 ± 1.02c

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SFAs saturated, MUFAs monounsaturated, PUFAs polyunsaturated fatty acids. Each value is the mean of three replicates ± SD. Values with the same letter in the same row showed insignificant differences (at P≤ 0.05).

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Fatty acid content (mg g-1 CDW)

In order to investigate the suitability of T. ellipticalipids as biodiesel feedstock,

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lipid classes at late exponential phase were determined (Figure 5). Total lipids

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represented 12.96 mg g-1 CDW; and neutral lipids represented 37 % of the total lipids

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(4.88 mg g-1 CDW). Polar lipids showed insignificant difference with neutral lipids (34 % 14

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of total lipids, P= 0.3086), while phospholipids recorded significant lower content (23 %

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of total lipids), Figure 5. Table 6 shows fatty acid composition of the different lipid

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fractions of T. ellipticaas well as the corresponding iodine value. Minor differences were

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found in the relative fatty acid content among different lipid classes. In all fractions,

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C16:0 and C18:1n-9 were predominant, being as high as 31 and 20 % of total fatty acids

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in neutral lipids, 26 and 24 % of total fatty acids in polar lipids and 28 and 26 % of total

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fatty acids in phospholipids, respectively. Saturated fatty acids largely prevail in all lipid

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classes (47, 46 and 39 % in neutral, polar and phospholipids, respectively). The recorded

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changes in different fatty acid classes for different lipid fractions were due mainly to the

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changes in C16:0 and C18:1n-9 metabolic pathways (Table 6). Iodine values recorded

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70.3, 128.8 and 162.1 g I2/100 g oil in neutral lipids, polar lipids and phospholipids,

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respectively (Table 6).

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Fig. 5. Total lipid content and different lipid classes (as mg g-1 CDW) of

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Tetraselmiselliptica at late exponential phase. 15

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Table 6.Fatty acid composition (as % of total fatty acids) and iodine value (as g I2/100 g

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oil) of the three different lipid classes of T. elliptica at late exponential phase.

Polar lipids

Phospholipids

C14:0

0.39

0.46

0.21

C16:0

30.55

26.04

27.81

C16:1n-9

0.33

0.16

0.13

C16:1n-7

1.45

1.89

0.19

C16:2

1.02

0.98

0.84

C16:3

1.04

0.94

0.62

C16:4n-3

9.70

C18:0

0.37

C18:1n-9

20.24

C18:2n-6

4.43

1.98

0.29

23.66

25.69

1.13

4.99

5.44

C18:3n-3

13.35

7.23

14.85

C18:3n-6

0.00

0.79

0.35

C18:4n-3

3.20

5.93

7.09

2.44

5.57

1.98

1.89

1.92

1.81

11.10

9.58

6.43

1.80

2.08

1.84

46.65

45.71

38.56

MUFAs

23.91

27.63

27.82

PUFAs

29.44

26.66

33.62

70.3 ± 1.9a

128.8 ± 3.6b

162.1 ± 2.8c

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Neutral lipids

C20:0 C22:0 C24:0

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SFAs

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C20:1n-9

Iodine value*

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Fatty acid proportion

Fatty acids

*Each value is the mean of three replicates ± SD. Values with different letters showed significant difference (P ≤ 0.05)

259 260 16

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261

4. Discussion Water characteristics of Bardawil Lagoon were studied by Ali et al. [19]. They

263

concluded that the salinity of Bardawil Lagoon is much higher than in the open sea as a

264

result of low rainfall (80-100 mm year-1) and high evaporation rate (1460 mm year-1).

265

The present study confirmed the high salinity of water at Bardawil Lagoon which

266

resulted in high sodium concentration with high visibility values. Water turbidity in lakes

267

is a result of organic turbidity (due to planktonic organisms) and/or mineral turbidity

268

(caused by clay and silt particles in suspension).Khalil et al. [28] reported relatively high

269

turbidity in Bardawil lagoon during winter and they attributed that to the continuous

270

mixing oflagoon water by the strong wind action which prevails in this season.

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Since microalgae are taxonomically diverse and not all strains are able to be

272

cultured, the efficient isolation and screening of microalgae is essential. Isolation of

273

suitable microalgae strains with high lipid content and high biomass production is the

274

bottlenecks of commercial biodiesel production. Lipid productivity is the product of lipid

275

content and biomass production, hence, it is dependent on both. However, lipid content

276

has not been shown to be a reliable indicator of lipid productivity, whereas a more

277

dominant correlation was observed between biomass and lipid productivity [14, 29].

278

Therefore, the isolated halophilicmicroalgae in the present work were selected not only

279

on the basis of high lipid and fatty acid content but also based on high biomass

280

production. The maximum biomass productivity (0.122 g CDW L-1d-1)was detected for

281

the chlorophyteT. elliptica , which is comparable to the finding of Griffiths and Harrison

282

[29] who reported average biomass productivity of T. suecica of 0.1 g L-1 d-1. However, it

283

is104 % higher than the finding of Moheimani[30] who reported biomass productivity of

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0.049 g L-1 d-1 for T. suecica cultivated on f/2 medium. In addition, Matsumoto et al. [31]

285

reported maximum biomass productivity of 0.053 g L-1 d-1 for the marine

286

chlorophyteChlorella sp. which is57 % lower than T. ellipticain our study. Moreover,

287

T.ellipticashowed high growth rates in the cost-effective Flory medium, which is the

288

most suited medium for industrial large scale cultivation [32].

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Tetraselmisellipticashowed the highest FAME content between all studied

290

species. Khatoon et al.[33] reported total lipid content for T.ellipticaof 14 % of CDW

291

which is in agreement with our finding (13.6 % of CDW). As a result of high biomass

292

productivity and high FAME content, T.ellipticashowed the highest FAME productivity

293

which represented 44 % higher than the finding of Moheimani[30] (2013) who reported

294

lipid productivity of 9.7 mg L-1 d-1 for T. suecica. Surprisingly, Francisco et al. [34] and

295

Abomohra et al. [14] reported that biomass productivity and lipid content are inversely

296

related. However, the present study showed that T.ellipticashowed the maximum biomass

297

productivity and highest FAME content among the studied species.

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Not all oils extracted from algae are suitable or compatible for use as

299

biodiesel[35]. The properties of biodiesel are determined mainly by its fatty acid methyl

300

esters profile [36-37]. UFAs with four or more double bonds are susceptible to oxidation

301

during storage, thus reduce the acceptability of microalgal oil for biodiesel production

302

[38]. Our results revealed that the SFAs and MUFAs content of T.ellipticaat late

303

exponential phase was 34 and 20 % of total fatty acids, respectively, with low

304

linolenicacid (C18:3) contents which agrees with the European standard specifications (≤

305

12 %, [38])and stimulate the oxidative stability of T.ellipticabiodiesel.

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For biodiesel production, neutral oils mainly constituted of triglycerides (TAGs)

307

can be easily converted into the corresponding FAMEs by transesterification. T. elliptica

308

showed neutral lipids as 51 % of total lipids which is higher than that reported

309

byBondioli et al. [2]who estimated27 % neutral lipids in T.suecica;and concluded that

310

neutral lipids could be increased to 53 % of total lipids under nitrogen starvation. In

311

addition, fatty acid composition of neutral lipid fraction (with 47 % SFAs and 29 %

312

PUFAs) is suitable for biodiesel production. Moreover, it is to mention that an iodine

313

value of 70.3 g I2/100 g oil was measured for the neutral lipid fraction, is significantly

314

lower than the limit established by the EN 14214 [38] of 120 g I2/100 g oil.

315

5. Conclusion

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The screening procedure provided useful ways to select microalgae, and is

317

recommended as an essential process for biodiesel feedstock production. Out of 21

318

isolated marine microalgae, 9 strains were characterized for high biomass and FAME

319

productivities. The best strain, based on FAME productivity, was T.ellipticawith high

320

biomass productivity (0.122 g CDW

321

136.46 mg g-1 CDW, respectively, and a corresponding FAME productivity of 14.1 mg L-

322

1

323

corresponding to a favorably high cetane number for biodiesel feedstock. Therefore,

324

T.ellipticarepresents an attractive alternative renewable biofuel feedstock. It is marine

325

microalga, which do not compete with food crops while increasing the environmental

326

cultivation possibilities. T.ellipticais undergoing on-going investigation to further

327

enhance lipid productivity to improve its feasibility as a feedstock for biodiesel

328

production.

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L-1

d-1), FAME and lipid contents of 77.36 and

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d-1. Furthermore, T.ellipticashowed a predominance of SFAs and MUFAs

19

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Acknowledgments

330

We thank Sigrid Mörke (Department of Cell Biology and Phycology, University of

331

Hamburg) for her excellent expert technical assistance. This work was supported by

332

grants from EgyptianMinistry of Higher Education and Scientific Research (to A.

333

Abomohra).

334

References

335

[1] B. Hankamer, F. Lehr, J. Rupprecht, J.H. Mussgnug, C. Posten, O. Kruse,

336

Photosynthetic biomass and H2production by green algae: from bioengineering to

337

bioreactor scale-up, Physiol. Plant. 131 (2007)10-21.

NU

SC RI PT

329

[2] P. Bondioli, L. Bella, G. Rivolta, G. Zittelli, N. Bassi, L. Rodolfi, D. Casini, M.

339

Prussi, D. Chiaramonti, M.R. Tredici, Oil production by the marine microalgae

340

Nannochloropsis

341

M33,Bioresour.Technol. 114(2012) 567–572.

F&M-M24

and

Tetraselmissuecica

F&M-

ED

sp.

MA

338

[3] C. Teo, M. Atta, A. Bukhari, M. Taisir, A. Yusuf,A. Idris, Enhancing growth and

343

lipid production of marine microalgae for biodiesel production via the use of

344

different LED wavelengths,Bioresour.Technol. 162(2014) 38–44.

CE

PT

342

[4] J. Pruvost, J.Legrand, H.Marec, B. Le-Gouic, B. Mirabella, B.Legeret, S. Bouvet, G.

346

Peltier, Y. Li-Beisson, S.Taha, H.Takache, Development and validation of a

347 348 349

AC

345

screening procedure of microalgae for biodiesel production: Application to the

genus of marine microalgae Nannochloropsis,Bioresour.Technol.177 (2015) 224–

232.

20

ACCEPTED MANUSCRIPT

351 352 353 354 355 356

[5] A. Demirbas, Use of algae as biofuel sources, Energy Convers. Manage. 51(2010) 2738-2749. [6] T.A. Milne, R.J. Evans, N. Nagle, Catalytic conversion of microalgae and vegetable

SC RI PT

350

oils to premium gasoline, with shapeselective zeolites, Biomass 21(1990)219–232. [7] M. Rohmer, The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants, Nat. Prod. Rep. 16(1999)565–574. [8] M. Matsumoto, H.Yokouchi, N. Suzuki, H.Ohata, T.Matsunaga,Saccharification of marine

microalgae

using

marine

bacteria

358

Appl.Biochem.Biotechnol. 105(2003)247–254.

for

ethanol

production,

NU

357

[9] R.C. Prince, H.S.Kheshgi, Photobiological production of hydrogen: potential

360

efficiency and effectiveness as a renewable fuel, Crit. Rev.Microbiol. 31(2005)19–

361

31.

MA

359

[10] Y. Chisti, Biodiesel from microalgae,Biotechnol.Adv.25 (2007) 294-306.

363

[11] A. Abomohra, M. El-Sheekh, D. Hanelt, Pilot cultivation of the chlorophyte

364

microalga Scenedesmusobliquus as a promising feedstock for biofuel, Biomass

365

Bioenerg. 64(2014) 237-244.

368 369 370

PT

CE

367

[12] R. Halim, B.Gladman, M.K.Danquah, P.A.Webley, Oil extraction from microalgae for biodiesel production,Bioresour.Technol.102 (2011)178-185. [13] M. El-Sheekh, A. Abomohra, D.Hanelt, Optimization of biomass and fatty acid

AC

366

ED

362

productivity of Scenedesmusobliquus as a promising microalga for biodiesel

production, World J.Microbiol.Biotechnol. 29(2013)915-922.

21

ACCEPTED MANUSCRIPT

[14] A. Abomohra, M. Wagner, M. El-Sheekh, D.Hanelt, Lipid and total fatty acid

372

productivity in photoautotrophic fresh water microalgae: screening studies towards

373

biodiesel production, J. Appl.Phycol. 25(2013)931-936.

SC RI PT

371

374

[15] L. Rodolfi, G.C.Zittelli, N.Bassi, G.Padovani, N. Biondi, G.Bonini, M.R. Tredici,

375

Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass

376

cultivation in a low-cost photobioreactor,Biotechnol.Bioeng. 102(2009) 100-112.

377 378

[16] X. Miao, Q. Wu, Biodiesel production from heterotrophic microalgal oil,Bioresour.Technol. 97 (2006)841-846.

[17] A. Widjaja, C.C. Chien, Y.H.Ju, Study of increasing lipid production from fresh

380

water microalgae Chlorella vulgaris,J. Taiwan Inst.Chem.Eng. 40(2009)13-20.

381

[18] M.T. Khalil, K.H.Shaltout, Lake Bardawil and zaranik protected area, Publication of

MA

382

NU

379

National Biodiversity Unit. No 15(2006) ISBN 977-17-4144-6. [19] M.H. Ali, M.F.Sayed, M.E.Goher, Studies of water quality and some heavy metals

384

in hypersaline Mediterranean Lagoon (Bardawil Lagoon, Egypt), Egypt.J. Aquatic

385

Biol.Fish. 10(2006)45-64.

PT

ED

383

[20] APHA/AWWA/WPCF,Standard Methods for the Examination of Water and

387

Wastewater, 16th ed. American Public Health Association, Washington(1985).

388

[21] R.L. Guillard, Culture of phytoplankton for feeding marine invertebrates. In W.L.

389

Smith,M.H. Chanley (Eds.) Culture of marine invertebrate animals, Plenum Press,

391 392

AC

390

CE

386

New York, USA (1975).

[22] A.A. Robert, Algal culturing techniques: traditional microalgae isolation techniques, Elsevier Academic Press, USA (2005)pp 83-98.

22

ACCEPTED MANUSCRIPT

[23] J. Folch, M. Lees, G.S. Stanley,A simple method for the isolation and purification of total lipids from animal tissues, J. Biol.Chem. 226(1957)497–509.

394 395

[24] E.G. Bligh, W.J. Dyer,A rapid method of total lipid extraction and purification, Can.

SC RI PT

393

J.Biochem.Physiol. 37(1959)911–917.

396 397

[25] D. Kaczmarzyk, M. Fulda, Fatty acid activation in Cyanobacteria mediated by acyl-

398

acyl carrier protein synthetase enables fatty acid recycling, Plant Physiol.

399

152(2010)1598–1610.

[26] M. Scharnewski, P. Pongdontri, G. Mora, M.Hoppert, M. Fulda, Mutants of

401

Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due

402

to interrupted fatty acid recycling, FEBS J. 275(2008)2765–2777.

NU

400

[27] S. Fakas,S. Papanikolaou, M.Galiotou-Panayotou, M.Komaitis, G. Aggelis, Lipids

404

of Cunninghamellaechinulata with emphasis to gama-linolenic acid distribution

405

among lipid classes, Appl.Microbiol.Biot. 73(2006) 676–683. [28]

M.

Khalil,

A.Saad,

macrobenthicinvertebrates

408

3(2013)1-8.

412 413

studies

on

of Bardawilwetland, Egypt, World Environment

[29] M.J. Griffiths, S.T. Harrison,Lipid productivity as a key characteristic for choosing

CE

411

Ecological

algal species for biodiesel production,J. Appl.Phycol. 21(2009)493–507. [30] N. Moheimani, Inorganic carbon and pH effect on growth and lipid productivity of

AC

410

T.Bedir,

PT

407

409

M.Fishar,

ED

406

MA

403

Tetraselmissuecica and Chlorella sp. (Chlorophyta) grown outdoors in bag

photobioreactors, J. Appl.Phycol. 25(2013)387–398.

414

[31] M. Matsumoto, H. Sugiyama, Y. Maeda, R. Sato, T.Tanak, T. Matsunaga, Marine

415

diatom, Navicula sp. strain JPCC DA0580 and marine green alga, Chlorella sp.

23

ACCEPTED MANUSCRIPT

416

strain

NKG400014

as

potential

sources

417

Appl.Biochem.Biotechnol. 161(2010)483–490.

for

biodiesel

production,

[32] S. Hindersin, M.Leupold, M.Kerner, D,Hanelt, Irradiance optimization of outdoor

419

microalgal cultures using solar tracked photobioreactors, Bioprocess Biosyst.Eng.36

420

(2013)345-355.

SC RI PT

418

[33] H. Khatoon, N. Abdu Rahman, S. Banerjee, N. Harun, S. Suleiman, N.Zakaria, F.

422

Lananan, S. Abdul Hamid, A. Endut, Effects of different salinities and pH on the

423

growth and proximate composition of Nannochloropsis sp. and Tetraselmis sp.

424

isolated from South China Sea cultured under control and natural condition,

425

Int.Biodeter.Biodegr. 95(2014) 11-18.

NU

421

[34] É.C. Francisco, D.B. Neves, E. Jacob-Lopes, T.T. Franco, Microalgae as feedstock

427

for biodiesel production: carbon dioxide sequestration, lipid production and biofuel

428

quality, J. Chem.Technol.Biotechnol. 85(2010)395–403. [35] M.C. Damiani, C.A.Popovich, D.Constenla, P.I.Leonardi, Lipid analysis in

ED

429

MA

426

Haematococcuspluvialis

431

feedstock,Bioresour.Technol. 101(2010)3801–3807.

434 435 436 437 438

its

potential

use

as

a

biodiesel

[36] C. Yoo, S. Jun, J. Lee, C.Ahn, H. Oh, Selection of microalgae for lipid production

CE

433

assess

under high levels carbon dioxide,Bioresour.Technol.101 (2010)71–74. [37] M. Battah, Y. El-Ayoty, A.Abomohra, S.Abd El-Ghany, A. Esmael, Effect of Mn2+,

AC

432

to

PT

430

CO2+ and H2O2 on biomass and lipids of the green microalga Chlorella vulgaris as a

potential candidate for biodiesel production, Ann.Microbiol. 65(2015)155–162.

[38] EN 14214, Automotive Fuels – Fatty Acid Methylesters (FAME) for Diesel Engines – Requirements and Test Methods(2008).

24

ACCEPTED MANUSCRIPT

[39] L. Provasoli, Media and prospects for the cultivation of marine algae, In A.

440

Watanabe, A. Hattori (Eds.) cultures and collections of algae. Proc. US-Japan Conf.

441

Hakone, Japan, (1968).

AC

CE

PT

ED

MA

NU

SC RI PT

439

25