- Email: [email protected]
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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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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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 ±
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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
242
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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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)
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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
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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)
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can be easily converted into the corresponding FAMEs by transesterification. T. elliptica
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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.
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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
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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
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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
3
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
26
characterized with respect to biomass and fatty acid productivities. Biomass productivity
27
as cellular dry weight (CDW), fatty acid content and, consequently, fatty acid
28
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
30
fractionation showed that total lipids represented 12.96 mg g-1 CDW and neutral lipids
31
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
33
total fatty acids in neutral lipids, 26 and 24 % of total fatty acids in polar lipids and 28
34
and 26 % of total fatty acids in phospholipids, respectively. This study demonstrates that
35
the halophilic microalga T. ellipticaisolated from hypersaline water is a promising species
36
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
45
world demand while mitigating climate change through CO2 sequestration and emissions
46
reduction, it is required to extendclean renewable fuels. Ironically, most renewable
47
energy initiatives were focused on electricity generation, while about two thirds of world
48
energy consumption is derived from liquid fuels [1]. Therefore, biomass-derived liquid
49
fuels are receiving greater attention; and with predictions that crude oil prices will reach
50
record
51
attention[2,3,4].Microalgae are unicellular or multicellularphotosynthetic microorganisms
52
that convert sunlight, water and carbon dioxide into biomass. They can be found in
53
diverse of environments and harsh conditions, living in saline or freshwater environments
54
[5]. Microalgae can provide several different types of renewable biofuels, including
55
anaerobic digestion of the algal biomass into methane, oil transesterificationinto
56
biodiesel, saccharification of carbohydrates into ethanol, cracking of hydrocarbons and
57
isoprenoidsinto gasoline and direct photobiologically synthesis of biohydrogen[6-11]. In
58
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
60
1970s several funded research programs in different countries were started to study the
61
efficiency of microalgae in biodiesel production [15].Microalgae have been cited as one
62
of the best non-edible feedstock for biodiesel compared to oleaginous crops, such as
63
soybean, rapeseed and oil palm. The priority of microalgae in biodiesel production is due
64
to 1) higher oil productivity which at least 15-20 times higher than conventional crops, 2)
65
high biomass production due to short doubling time, 3) does not necessarily require
highs,
algal
based
biofuels
are
gaining
widespread
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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
68
previous advantages, marine microalgae have additional preference that they don’t
69
compete for freshwater.
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Bardawil Lagoon is one of the five northern lakes in Egypt. It is bordered from the
71
south by a sand dune belt and from the north by a convex sand barrier that separates it
72
from the Sinai Mediterranean coast (Figure 1). The area of that lake is about 685 km2 and
73
extends for a distance of 80 km with maximum width of 20 km and maximum depth of 3
74
m [18]. The water temperature values fluctuated between a minimum value of 11.6 ºC
75
during January to a maximum value of 33.2 ºC in July, with annual mean of 21.5 ± 6.5 ºC
76
and has salinity much higher than that of the open sea [19]. The key technical challenge
77
in biodiesel production by microalgae is to identify a strain with the highest growth rates
78
and oil contents with suitable fatty acid profile. The present study examines the efficiency
79
of marine microalgae isolated from Bardawillagoon as a source of biomass and biodiesel
80
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
84
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
88
microalgal strains in the present study.Microalgae were isolated from three water samples
89
collected during April 2013 from Marsa El-Nasr (N 31º 05\ 26\\, E 32º 52\ 17\\),
90
Bardawillagoon, Sinai Peninsula, Egypt. The samples were collected in plastic bottles
91
and brought to the laboratory shortly after collection. Temperature, pH, water depth,
92
turbidity, salinity and water transparency were measured in the field. Water transparency
93
was measured using Secchi disc of 25 cm in diameter. Some water parameters were
94
measured directly after collection (Na, K, Ca, Mg, total N, total P, Mg, K, Na and Ca).
95
Atomic absorption spectrophotometer (Shimadzu AA-6300) was used for determination
96
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
101
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
104
sub-culturing as previously described by Robert [22]. A total of 21 marine microalgal
105
strains were isolated from the collected water samples in unialgal cultures, however nine
106
strains were chosen after the first selection (Figure 2) depending on their relatively higher
107
growth. Selected strains were identified using their morphological features and deposited
108
in Tanta University Culture Collection, Tanta, Egypt.
109
2.3. Growth and biomass assay
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For selection of media providing the best growth, each strain was individually
111
cultivated in 100mL Erlenmeyer flasks at 20 ± 1 °C. For this purpose, six different
112
marine media were used (Table 1). The Flory medium was prepared by dissolving of 2 g
113
of N-free Flory Basic Fertilizer 1 (Euflor, Germany) and 810 mg potassium nitrate as
114
nitrogen source in 1 L of natural seawater. Optical density (OD540) was measured after 12
115
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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119 120 121
BMS
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A certain volume of exponentially growing microalga,precultured in 1 L
123
Erlenmeyer flasks on the best medium, was inoculated in 300 mL of the corresponding
124
medium in Kniese tubes [13] at an initial OD540 of 0.06. Sterile filtered air enriched with
125
1.5 % (v/v) CO2 was continuously applied to all cultures. Algal growth was monitored
126
using the optical density of the culture at 540 nm (OD540) and by determination of algal
127
cellular dry weight (CDW). Biomass productivity was calculated according to Abomohra
128
et al. [14].
129
2.4. Lipid extraction
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Extraction of lipids was performed using chloroform:methanol (2:1)according to
131
Folch et al. [23]. Lipid extracts were dried under a stream of argon. The pre-weighed
132
glass vials containing the lipid extracts were dried at 80 °C for 30 min, cooled in a
133
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
137
were collected at times specified. Lipids were extracted following the method of Bligh
138
and Dyer [24]. Prior to extraction, trinonadecanoylglycerolwas added to the samples as
139
internal standards. To determine the fatty acid profiles, esterified fatty acids from the
140
extracts of intracellular lipids were converted to fatty acid methyl esters
141
(FAMEs)bytransmethylationas described previously [25-26]. FAMEs were subjected to
142
analysis by GC (Varian 3900 GC-system equipped with a Varian capillary column, Select
143
Fame, 50 m length and 0.25 mm internal diameter); and FAME productivity was
144
calculated according to Abomohra et al. [14].
145
2.6. Fractionation of lipids
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Lipid fractionation was performed to the total lipid extract of the promising
147
microalga according to the method described by Fakas et al. [27]. Briefly, 50 mg of total
148
lipid extract were dissolved in 1 ml chloroform, and fractionated using a column (25 mm
149
× 100 mm) containing 1 g silicic acid activated by heating overnight at 80 °C. Successive
150
applications of 100 ml dichloromethane, 100 ml acetone and 100 ml methanol produced
151
fractions containing neutral lipids, polar lipids and phospholipids, respectively. Lipid
152
fractions were collected respectively and carefully weighed after solvent removal.
153
Individual fatty acid proportions were measured in each lipid fraction using GC as
154
described in the previous chapter. Degree of unsaturation (iodine number) for each
155
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.
159
The statistical analyses were carried out using SAS (v 6.12). Data obtained were analyzed
160
statistically to determine the degree of significance using one-way and two-way analysis
161
of variance (ANOVA) at probability level P≤ 0.05.
162
3. Results
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Three water samples were collected around the location known as Marsa El-Nasr
164
at Bardawil lagoon. Water at the collection sites showed neutral pH value of 7.43 with
165
average temperature of 19.9 °C and 39.4 ‰ salinity (Table 2). Macronutrients analysis
166
indicated relatively high concentrations of sodium (11538 mg L-1) followed by
167
magnesium (1264 mg L-1), while total nitrogen and total phosphorus represented 75.6 and
168
0.068 mg L-1, respectively (Table 2).
169
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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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
172
relatively high growth which was noticed during isolation steps (first selection). In order
173
to select a media that facilitates high biomass productivity in batch cultures, each strain
174
was cultivated in six different media, and the growth was monitored by OD540 after 12
175
days of incubation (Table 3). Chlorella vulgaris,andTetraselmisellipticashowed
176
maximum growth on FM, while Cyclotella sp., Dunaliellasalinaand Navicula sp.showed
177
maximum growth on SES. F/2 was recorded as the best medium for growth of
178
Naviculamolli, Chlorella sp.1,Chlorellasp.2 and Pinnularia sp.(Table 3). Consequently,
179
only the medium providing highest growth for each strain was used for further
180
experiments.
181
Table 3. Growth of isolated microalgae (as OD540) cultivated in different growth media
182
for 12 days.
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Growth (OD540)
Microalgae SES
F/2
MAS
ES
FM
ED
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.
183 184 185
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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
187
sp.;and D. salinato 16 days for Navicula sp. (Table 4). T. elliptica showed highest
188
biomass productivity of 0.122 g CDW L−1d−1 which was 31, 50, 52 and 54 % higher than
189
Cyclotella sp.,Chlorella sp.1, Chlorella sp.2 andPinnularia sp., respectively. While C.
190
vulgarisshowed the lowest biomass productivity of 0.016 g CDW L−1d−1 (87 % lower
191
than T. elliptica, Table 4).
192
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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Organisms
12
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
8
0.085 ± 0.003d
0.220 ± 0.057
0.827 ± 0.056
10
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
14
0.056 ± 0.002e
Navicula sp.
SES
0.388 ± 0.027
0.772 ± 0.041
16
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
196
PT
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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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193 194 195
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
197
species, which showed significant differences between studied species (one way
198
ANOVA, P= 0.0001 for both). Overall, T. elliptica showed the highest significant (one
199
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
201
CDW). As a result of high FAME content and high biomass production of T. elliptica, it
202
showed the highest FAME productivity (14.1 mg L-1 d-1) which was 75 and 74 % higher
203
than Chlorella sp.1 and Cyclotella sp. , respectively (Figure 3).
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204
Fig. 3.FAME productivities (grey bars) and FAME content (black dots) of different
206
isolated microalgae during the exponential phase. Error bars show the SD of three
207
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
210
mg g-1 CDW), it showed the lowest FAME productivity (0.361 mg L-1 d-1) due to its low
211
biomass production (0.024 g L-1 d-1).Therefore, T. elliptica (Figure 4) was selected
212
(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)
216
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
219
early exponential phase, middle exponential phase and late exponential phase.
220
Insignificant changes (one way ANOVA, P=0.8213) were recorded in total fatty acid
221
content from early exponential phase to late exponential phase (Table 5). However
222
transition from early to late exponential phaseresulted in significant increase in the
223
proportion of saturated and monounsaturated fatty acids by 31 and 52 %, respectively,
224
with consequent 41 % significant decrease in polyunsaturated fatty acids (Table 5). The
225
dominant fatty acids were 16-carbon and 18-carbon fatty acids, which showed
226
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
231
phases. Fatty acids
236
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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Early exponential phase
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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0.83a
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,
237
lipid classes at late exponential phase were determined (Figure 5). Total lipids
238
represented 12.96 mg g-1 CDW; and neutral lipids represented 37 % of the total lipids
239
(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 %
241
of total lipids), Figure 5. Table 6 shows fatty acid composition of the different lipid
242
fractions of T. ellipticaas well as the corresponding iodine value. Minor differences were
243
found in the relative fatty acid content among different lipid classes. In all fractions,
244
C16:0 and C18:1n-9 were predominant, being as high as 31 and 20 % of total fatty acids
245
in neutral lipids, 26 and 24 % of total fatty acids in polar lipids and 28 and 26 % of total
246
fatty acids in phospholipids, respectively. Saturated fatty acids largely prevail in all lipid
247
classes (47, 46 and 39 % in neutral, polar and phospholipids, respectively). The recorded
248
changes in different fatty acid classes for different lipid fractions were due mainly to the
249
changes in C16:0 and C18:1n-9 metabolic pathways (Table 6). Iodine values recorded
250
70.3, 128.8 and 162.1 g I2/100 g oil in neutral lipids, polar lipids and phospholipids,
251
respectively (Table 6).
252
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253
Fig. 5. Total lipid content and different lipid classes (as mg g-1 CDW) of
254
Tetraselmiselliptica at late exponential phase. 15
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255
Table 6.Fatty acid composition (as % of total fatty acids) and iodine value (as g I2/100 g
256
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*
257 258
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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,
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Photosynthetic biomass and H2production by green algae: from bioengineering to
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bioreactor scale-up, Physiol. Plant. 131 (2007)10-21.
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[2] P. Bondioli, L. Bella, G. Rivolta, G. Zittelli, N. Bassi, L. Rodolfi, D. Casini, M.
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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-
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sp.
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