Carotenoid and fatty acid compositions o

Carotenoid and Fatty Acid Compositions of an Indigenous Ettlia texensis Isolate Chlorophyceae Under Phototrophic and Mixotrophic Conditions Arzu Yıldırım&Zeliha Demirel&Mügeİşleten-Hoşoğ

Carotenoid and Fatty Acid Compositions of an Indigenous Ettlia texensis Isolate (Chlorophyceae) Under Phototrophic and Mixotrophic Conditions

Arzu Yıldırım&Zeliha Demirel&Mügeİşleten-Hoşoğlu&

İsmail Hakkı Akgün&Sevde Hatipoğlu-Uslu&

Meltem Conk-Dalay

Received: 24 July 2013 / Accepted: 8 October 2013

# Springer Science+Business Media New York 2013

Abstract Ettlia oleoabundance (formerly known as Neochloris oleoabundance) is an attractive candidate for biodiesel production because of its high lipid accumulation, and it’s taking the majority of the attention among the strains of Ettlia genus; however, potential of the other genus members is unknown An indigenous strain from Salda Lake (South West Turkey) identified by 18S rDNA sequencing as Ettlia texensis (GenBank accession no: JQ038221), and its fatty acid and carotenoid compositions under phototrophic and mixotrophic conditions was investigated to evaluate the potential of the strain for commercial uses A threefold increase was observed in total lipid content (total fatty acids; from 13 % to 37 %) in mixotrophic culture respect to the phototrophic growth conditions The oleic acid (C18:1) and alpha-linolenic acid (18:3) were the major unsaturated fatty acids accounting for 40 % and 13.2 % of total fatty acids

in mixotrophic culture, respectively Carotenoid analyses of the mixotrophic culture revealed the metabolite canthaxanthin, a commercially valuable carotenoid used mainly for food coloring, was the major constituent among other pigments The possible use of E texensis in biotechnological applications is discussed

Keywords Carotenoid Cell disruption Ettlia texensis Fatty acid Molecular identification Neochloris

Abbreviations

TAGEM Turkish General Directorate of Agricultural Research and Policy

TUBITAK The Scientific and Technological Research Council of Turkey

PHOT Phototrophic culture conditions

EGEMACC The Microalgae Culture Collection of Ege University

DOI 10.1007/s12010-013-0599-y

A Y ıldırım (*):Z Demirel:M İşleten-Hoşoğlu:İ H Akgün:S Hatipo ğlu-Uslu:M Conk-Dalay Engineering Faculty, Department of Bioengineering, Ege University, 35100 Bornova İzmir, Turkey e-mail: arzuyildirim78@gmail.com

NCBI National Center for Biotechnology Information

MEGA Molecular Evolutionary Genetics Analysis

U-HPLC Ultra high-performance liquid chromatography

Introduction

The use of microalgae in biotechnology has the advantage on other production systems such

as simple growth requirements and short life cycles with respect to animal and plant cell cultures [1] Therefore, there is an increasing effort to use microalgae as a natural source of high-value compounds like pigments, polyunsaturated fatty acids, and polysaccharides [2] During recent years, the importance of microalgae has also increased with the idea of using them as a renewable energy source like ethanol, hydrogen, and biodiesel production [3] Many algal species were evaluated for their ability to produce high levels of lipids, particularly the genus Chlorella, Dunaliella, and Neochloris were found to be important for their high lipid accumulation capacities in different cultivation conditions [4]

The green algae Neochloris was first described [5] under Chlorococcaceae family However, in time, the members of this genus either regrouped or renamed by different researchers [6] Today, the genus is represented by 18 species under the names of Neochloris, Ettlia, Parietochloris, and Pseudoneochloris depending on their uni- or multi-nuclear structure, wall formation around zoospores, and the orientation of basal bodies [7]

In order to overcome the complexity in morphological identifications, 18S rDNA sequence was frequently used for the molecular identification of eukaryotic microalgae which is giving the opportunity to identify the species of interest rapidly and more precisely [8,9] Neochloris oleoabundance, recently called Ettlia oleoabundance is a freshwater species that produces up to 80 % triglycerides of its total lipids, and most of its fatty acids are

in saturated form in the range of 16–20 [3] Among the species of Ettlia genus, E oleoabundance is widely studied for enhancing its lipid content in different growth conditions [10–12]; however, there is limited information about other species of the genus on growth characteristics and production of metabolites for the utility in biotechnology field [13]

In this study, we focused on an indigenous microalgal isolate from Salda Lake (Turkey) which was characterized by morphological methods as Neochloris sp Molecular studies revealed the strain as Ettlia texensis based on 18S rDNA region Evaluation of growth, lipid production, and carotenoid and fatty acid composition of the cells cultivated under phototrophic (PHOT) and mixotrophic (MIX) conditions were accomplished, and different cell disruption techniques were applied to make an efficient extraction process for both carotenoid and lipid determinations

Materials and Methods

Algal Strain and Culture Conditions

The material strain of this study was obtained from The Microalgae Culture Collection of Ege University, Turkey (EGEMACC-68), which was originated from Salda Lake (Turkey), and named as Neochloris sp before molecular identifications Two cultures were grown in a modified bold basal medium (BBM) [14] under continuous aeration (1 min/L air pump) and

Appl Biochem Biotechnol

100μmol×phot/m2×s of continuous light (day light fluorescent lamp) at a temperature of 21±1 °C After 23 days of cultivation, PHOT culture was harvested by centrifugation (1,500×g for 5 min) and washed twice with demineralized water to obtain the algal paste which was stored at−20 °C until lyophilization The biomass was lyophilized using a freeze-dryer (Christ, Alpha 1–2 LD plus, Germany) and dried at −52 °C and 0.030 bar for 7 h, then finally dried at−55 °C and 0.021 bar for 3 h The dried biomass obtained after freeze-drying was stored in air-tight containers at−20 °C Second culture was grown for additional 10 days

in nitrate deficient BBM containing 6 g/L glucose and 45 mM acetate buffer in order to obtain MIX material, which was harvested and freeze-dried as well

The cell counts were quantified by using a Neubauer hemocytometer The optical density of the culture was evaluated by using a spectrophotometer (Ultraspec 1100 pro),

at λ=685 nm Morphological characterization of the strain was performed by light microscopy (Olympus)

DNA Isolation and PCR Analysis

Total genomic DNA from the freshwater microalgae was isolated with the ZR Fungal/Bacterial DNA MiniPrep (ZymoResearch) following the manufacturer’s instructions and stored at −20 °C

A universal primer sequence for the 18S gene was amplified by polymerase chain reaction (PCR) using the primers (For; 5'-TGGTTGATCCTGCCAGTAG-3', Rev; 5'-TGATCCTTCC GCAGGTTCAC-3') and PCR conditions of [8] Analysis was performed in BioRad MyCycler thermal cycler in Helix Amp™ Hypersense DNA polymerase (Nannohelix) by SSU primer pairs following the manufacturer’s instructions 18S rDNA amplification was performed in 50 μL reactions using primers SSU1 and SSU2, with an initial denaturation step at 95 °C for 2 min followed by 35 cycles of 95 °C for 30 s, 54 °C for 40 s and 72 °C for 40 s, and a final extension step at 72 °C for 5 min The PCR products were analyzed by 1 % agarose gel electrophoresis in Tris–boric acid–EDTA (TBE) buffer 1×, at 5 V/cm and stained with SYBR safe and visualized under UV illumination

Phylogenetic Analysis

Sequence analysis of the PCR amplicons were performed by Izmir Institute of Technology, Biotechnology and Bioengineering Central Research Laboratories, Turkey, with the Applied Biosystems 3130XL (16-capillary) Genetic Analyzer The sequence data were submitted into the NCBI database with the GenBank accession number JQ038221 and aligned with the reference sequences retrieved from GenBank using the ClustalW program version 4.0 in MEGA 5 [15] Phylogenetic tree was constructed with neighbor-joining, maximum parsimony, and maximum likelihood algorithms using CLC Sequence Viewer

Cell Disruption and Carotenoid Extraction

Since Ettlia texensin has a strong cell wall structure resisting the extraction process, cell disruption technique was optimized before continuing further lipid and carotenoid extractions Spectrophotometric measurement of total carotenoid content was evaluated to compare the efficiency of different disruption methods Total carotenoids were extracted with acetone/water (80:20 v/v) and measured by spectrophotometry according to the reference [16] The 0.01 g of lyophilized cell powders from PHOT and MIX cultures were disrupted by the following techniques: metallic beads, ceramic beads, cellulase enzyme, and sonication All extractions were repeated three times

Bead Applications Cell powder was ground in 2-mL Eppendorf tube by vortexing at maximum for 3 min in the presence of metallic beads and 3 min more after the addition

of 2 mL of acetone/water (80:20v/v) Metallic beads were removed from the tube by a magnetic bar rod; remaining solution was centrifuged at 5,000 rpm for 5 min at MiniSpin Centruge (Eppendorf) White pellet was discarded, and supernatant was measured atλ=470,

662, and 646 nm at Helios Alpha UV/Visible Spectrophotometer Same procedure was applied for the ceramic beads except, in this case, the mixed solution was separated from the beads by a micropipette to a new Eppendorf tube for the following steps

Sonication Both samples were sonicated by a sonicator probe (Bandelin Sonoplus 2070) with a 45 % power, 9 cycles for 15 min in 15 mL falcon tubes embedded on ice Pellet was subjected to carotenoid extraction as indicated above

Enzyme Treatment The 0.1 g of cellulase enzyme (Sigma Aldrich, Cat No: 22178) was added into 10 mL of 0.1 M sodium phosphate buffer (pH 7.0) The 0.01 g of PHOT and MIX dry cells were dissolved in 1 mL of cellulase enzymes buffer and incubated at 40 °C for 6 h The enzyme–microalgal mixtures were gently stirred at the end of incubation After enzyme hydrolysis, samples were separated by centrifugation (11.952×g for 10 min) Supernatant was discarded, and the pellet was used for carotenoid extraction

Sample Preparation for U-HPLC

Pigment extraction for the U-HPLC analysis was performed with the metallic beads according

to the results of the cell disruption experiments The 0.1 g of lyophilized material each from PHOT and MIX cultures was vortexed at maximum speed in the presence of beads for 5 min in

a 15 mL falcon tube and an extra 5 min after adding Acetone (Merck, Germany) After the removal of the metallic beads, the mixture was centrifuged for 5 min at 5,000 rpm in Hettich Universal 32 R Centrifuge Solvent phase was dried in SpeedVac Concentrator vacuum evaporator (Thermo Scientific) at 35 °C, and the residue was dissolved in 2 mL of acetone Carotenoid Determination by U-HPLC Analysis

Methanol was purchased from Merck (Germany) and ultrapure water used in the analysis was obtained from in-house ultra-pure water system (Sartorius Arium 611, Sartorius-Stedim, Gottingen, Germany) Lutein,β-carotene, zeaxanthin, chlorophylls a and b (Sigma-Aldrich, USA), and violaxanthin (ChromaDex, USA) were used as authentic standards Analyses were performed with a Thermo Accela U-HPLC system equipped with an autosampler, diode array detector, and quaternary pump (Thermo Fisher Scientific Inc., Massachusetts, USA) Thermo Hypersil RP C-8 column (100 × 2.1 × 3 μm) was used for analysis Separations were carried out using following solvents: ultrapure water (A) and methanol (B); and gradient elution was performed from 0 min 20A/80B, in 5 min to 0 A/100B, and kept at 0A/100B for 25 min Prior to the next injection, the column was equilibrated for

5 min with the starting conditions Flow rate was 0.800 mL/min; column temperature was

40 °C, and detection was performed at 450 nm

Total Lipid Content and Fatty Acid Composition

Lipids were extracted from lyophilized algal biomass by a modified method [17] Freeze-dried cells (100 ± 1.5 mg) were weighed accurately into a 15-mL centrifuge tube and

Appl Biochem Biotechnol

disrupted by metallic beads as indicated above For extraction, 3 mL chloroform/methanol (2:1) containing 1.0 mg/mL nonadecanoic acid (19:0) and 0.5 mg/mL BHT was used, and the tube was shaken gently overnight at room temperature After centrifugation at 1,500×g for 5 min, the supernatant containing the extracted oil was stored at 4 °C until analysis The extract was evaporated in a water bath (30 °C) using a rotary evaporator (Stuart, RE300, UK) to remove solvents The final lipid concentration was determined gravimetrically Fatty acids were analyzed by gas chromatography after direct transmethylation with hydrochloric acid in methanol with small modifications [18] The fatty acid methyl esters were extracted with n-hexane and analyzed by Agilent 7890 gas chromatography equipped with a flame-ionization detector and a Supelco sp-2380 A capillary column (60 m×250μm×0.2 μm) with helium as a carrier gas at a flow rate of 0.7 mL/min One-microliter sample was injected in the split (20:1) injection mode The inlet and detector temperatures were 260 °C, and the oven temperature was programmed at an initial temperature of 100 °C, then increased at 10 °C/min interval to 250 °C, and held there for 3 min Fatty acid methyl esters were identified by chromatographic comparison with authentic standards (Sigma Chemical Co., St Louis, MO)

Results

Morphological and Molecular Analysis

Based on the observations from light microscopy, the morphological features of the strain were as follows: The vegetative cells were single and ellipsoidal with a cup-shaped parietal chloroplast and a pyrenoid Adult cells were 6–25 μm, and young cells were 5 μm in diameter The maximum cell concentration of 9.6×105cell/mL was obtained in the BBM for

23rdday of cultivation under PHOT condition Culture was grown until the late logarithmic phase of growth (Fig 1) Doubling time of the culture was calculated approximately 2.6 days

For molecular analysis, DNA was isolated and visualized on 0.8 % agarose gel, and PCR products were separated on 1 % agarose gel at 5 V/cm (Fig.2)

BLAST search on NCBI-nucleotide database (http://www.ncbi.nlm.nih.gov/BLAST) resulted in the highest similarity to E texensis, which is a taxonomic synonym of Neochloris

texensis, with the identity of 94 % only Phylogenetic tree of sequences from all major lineages of the Chlorophyta is shown in Fig.3

Carotenoid Composition

Different disruption techniques resulted in different levels of carotenoid contents Bead extractions clearly provided better results than sonication and enzyme treatments (Fig.4) The highest total carotenoid amount (Table1; 7.36±0.6 mg/g in PHOT and 1.61±0.1 mg/g

in MIX culture) was obtained with metallic beads

Carotenoid content of the PHOT culture was almost four times higher than the MIX cells U-HPLC analyses revealed that carotenoid profile of PHOT was mostly consisting of lutein (Fig 5, A3 and A6) However, in the case of MIX, a reduction in lutein content was

Fig 2 DNA and PCR products

from Neochloris sp.; Lane 1:

size markers (100 bp DNA ladder

BioLabs, England), Lane 2: DNA

isolation of Neochloris sp.,

Lane 3: 18S rDNA (primers

SSU1-SSU2)

Appl Biochem Biotechnol

observed, whereas appearance of secondary carotenoids was noteworthy (Fig.6, B) The peak 5 (Fig.6, B5) was predicted to be canthaxanthin according to its retention time and absorption maxima (476 nm in acetone), which was significant for the MIX culture Fatty Acid Composition

The total lipid content of E texensis cultured in MIX conditions rose to 37 %, which was almost threefold higher than the PHOT conditions (13 %) Similar to lipid content difference, there were quantitative variances in fatty acid compositions of PHOT and MIX cells As shown in Table2, C16 and C18 fatty acids were the major fractions, which accounted for more than 97 % of total fatty acids (TFA) C16:0, C18:1, C18:2, and C18:3 n-6 were the major fractions The cells also contained small amounts of C14:0, and other C16, C18 fatty acids The oleic acid (C18:1) and alpha-linolenic acid (18:3) were the principal unsaturated fatty acids accounting for 40 % and 13.2 % of TFA, respectively, in stressed (MIX) cells Linoleic acid (C18:2) was the third most abundant unsaturated fatty acid (12.8 %) The most abundant saturated fatty acid was palmitic acid (C16:0), which constituted 17.0 % of TFA in MIX cells MIX cultures of E texensis produced a greater percentage of C18:1 fatty acids with respect to PHOT conditions (18.6 %)

Discussion

Cell Morphology and Molecular Data

Despite morphological similarities of Ettlia species to the traditional cell structure of Neochloris, ultrastructural and molecular studies reveal several dissimilar phylogenetic

Fig 4 Effect of different disruption methods on total carotenoid content under PHOT and MIX conditions

Table 1 Carotenoid content of E.

texensis under PHOT and MIX

conditions

E texensis Biomass Total Carotenoid Lutein

mg l−1 mg l−1 mg/g mg l−1 mg/g PHOT culture 463 3.4 7.36 0.104 0.23 MIX culture 207 0.33 1.6 0.0102 0.05

lineages Members of the genus Neochloris, which belong to the order Sphaeropleales, are characterized by their multinucleate structure, and they reproduce by releasing flagellated asexual spores, called zoospores [6,19] However, species of the Ettlia genus, which is recently used as synonym of some Neochloris species, belong to the order Volvocales in Chlorophyceae class; they have uninucleate cells and zoospores showing thin-walled cells [6,20] In this study, since the isolate did not form flagellate spores, we were able to define its phylogenetic position within the Chlorophyta only at the class level by morphological observations The phylogenetic analysis based on 18S rDNA (SSU; small subunit rDNA gene) for Neochloris species was previously studied [8] Using the same primer pairs (SSU1 and SSU2), the analyses of 18S rDNA sequences clearly indicated that the strain showed a similar phylogenetic relationship with the species of Scenedesmus and Ettlia within the Sphaeropleales order, and due to its close relation, it was named as E texensis

Effect of Disruption Techniques

Algae are known to possess multiple layers in their cell walls [21] Most of the microalgal cell walls encompass cellulose, and some species have an additional tri-laminar sheath, a material that is known for its resistance to disruption [22,23] From biotechnological point

Fig 5 A: Carotenoid profile of PHOT culture; 1 (A-1) violaxanthin; 2 (A-2) unknown; 3 (A-3) lutein derivative; 4 (A-4) unknown; 5 (A-5) zeaxanthin; 6 (A-6) lutein; 7 chlorophyll b; 8 chlorophyll a

Appl Biochem Biotechnol

of view, it is an important step to get rid of this cell wall to reach the intracellular compounds

of interest efficiently In our study, different cell disruption techniques were applied to see their effects on the extraction of carotenoid-type metabolites in E texensis cells Cellulase enzyme was the less efficient application compared with the other disruption methods for carotenoid extraction Mendes-Pinto et al [24] used an enzyme mixture consisting of protease K and driselase for extraction of the pigment astaxanthin from Haematococcus pluvialis; however, a reduction in total carotenoid yield was reported respect to the mechanical disruption methods, coinciding with our results Cellulase enzyme was

Table 2 Comparison of fatty acid composition of E texensis under PHOT and MIX conditions

E texensis Fatty acid composition (%TFA)

C16:0 C18:0 C18:0(t) C18:1 C18:2 C18:3 PHOT culture 20.5±0.2 4±0.1 12.1±0.1 18.6±0.2 14.5±0.1 24.2±0.2 MIX culture 17.0±0.1 6.5±0.2 4.0±0.1 40.0±0.2 12.8±0.1 13.2±0.3 Fig 6 B Carotenoid profile of MIX culture; 1 (B-1) violaxanthin; 2 (B-2); 3 (B-3) unknown; 4 (B-4) lutein; 5 (B-5) canthaxanthin (tentative −A max , 476 nm); 6: chlorophyll b; 7 chlorophyll a

recommended to be used in combination with pectinase for more effective extraction of carotenoids [25]; nevertheless, the enzymatic approach for carotenoid extraction was not suitable due to both low level of extracts and high costs of the treatment Metallic and ceramic beads were more effective to break the cell wall; thus, the cell content was fully acquired into the solvent during the following steps in a shorter time As a similar approach, Pirastru et al [26] used glass beads in order to destroy the wall of Scenedesmus sp cells for the extraction of carotenoids Among these three materials, metallic beads were the most effective due to the ease of removal from the solvent simply by a magnetic device Carotenoid Composition

In further analysis of carotenoid composition in PHOT and MIX cells by U-HPLC method, two main pigments named lutein and canthaxanthin were notable Takaichi [27] reviewed the distribution and function of carotenoids in different algae groups and reported that the main carotenoids observed for Chlorophyceae class were α- and β-carotene, lutein, zeaxanthin, violaxanthin, and neoxanthin Moreover, in certain stress conditions, some specific xanthophylls such as astaxanthin, canthaxanthin, and echinenone were produced in some green algae species In our study, the amount of total carotenoids in E texensis was 7.3 and 1.6 mg/g

in PHOT and MIX, respectively, which was considerably low compared with other species recommended for carotenoid production The amount of lutein in PHOT was 0.23 mg/g (Table1), which was not comparable with the amounts that del Campo et al [28] reached with Mureillopsis sp (5.55 mg/g) This type of poor content was previously reported [29,30] for Scenedesmus vacuolatus, which has molecular similarity to E texensis (Fig.3)

Total Lipid Content and Fatty Acid Composition

It’s been reported [10] that the nitrogen deficiency would result in more metabolic flux generated from photosynthesis to be turned to lipid accumulation in Neochloris oleoabundans The reason might be that the formation of essential cell structures including proteins and nucleic acids is suppressed under nitrogen deficiency or limitations Therefore, the major part of carbon fixed is converted into carbohydrate or lipid [10] The total lipid content values between 25 % and 54 % of dry weight (w/w) were common during nitrogen-depleted growth of E oleoabundans in freshwater conditions In this study, the total lipid content was found 37 % of dry weight under nitrogen-depleted, carbon-supplemented MIX conditions, supporting the previous studies

Fatty acid compositions of PHOT and MIX cells showed differences in E texensis as described by [31]; the most abundant fatty acids under nitrogen depletion were C16:0 and C18:1 This result was also confirmed in our study by observing a twofold increase of the relative content of C18:1 fatty acid under nitrogen depleted conditions The percentage of unsaturated fatty acids, especially C18:3, decreased in the stressed cells Lipids from E texensis seem to be feasible for biodiesel production because of high proportions of C16:0, C18:1, and C18:2, predominant components (C16–C18) of biodiesel [32] Also, compared with PHOT culture, MIX cells attained a much higher amount of oleic acid, which balances oxidative stability and low-temperature properties well and promotes the quality of biodiesel [33] Relationship Between Lipid and Carotenoid Production

Recent studies suggested that the carotenoid formation could be influenced by nitrogen limitation and also using some organic nutrients such as acetate and glucose in general [30,34] These

Appl Biochem Biotechnol