Fatty Acid Methyl Esters of the Aerophytic Cave Alga Coccomyxa subglobosa as a Source for Biodiesel Production

: The microscopic alga Coccomyxa subglobosa , collected from the Głowoniowa Ny˙za Cave (Tatra Mountains, Poland), is a source of fatty acids (FAs) that could be used for biodiesel production. FAs from subaerial algae have unlimited availability because of the ubiquity of algae in nature. Algal culture was carried out under laboratory conditions and algal biomass was measured during growth phase, resulting in 5 g of dry weight (32% oil). The fatty acid methyl ester (FAME) proﬁle was analyzed by means of gas chromatography–mass spectrometry (GC–MS). The presence of lipids and chloroplasts in C. subglobosa was demonstrated using GC–MS and confocal laser microscopy. Naturally occurring FAMEs contained C 12 –C 24 compounds, and methyl palmitate (28.5%) and methyl stearate (45%) were the predominant lipid species. Aerophytic algae could be an important component of biodiesel production, as they are omnipresent and environmentally friendly, contain more methyl esters than seaweed, and can be easily produced on a large scale.


Introduction
Biofuels produced from plants and animal waste reduce the necessity for petroleum, coal, and natural gas use and they are also an environmentally sustainable alternative that allows for reductions in global pollutants. Microalgae can produce third-generation biodiesel, because of the high content of lipid reaching up to 40% lipid by weight [1]. Biodiesel is now typically commercially produced from vegetable and soybean oils. However, oilseed crops cannot displace conventional transport fuels such as gasoline and diesel. Fakhry and Maghraby noted that the main environmental benefits of biodiesel depend on its renewability, lack of toxicity, and biodegradability, as well as the fact that its combustion produces fewer harmful gaseous emissions, such as sulphur oxide [2]. Biodiesel is composed of long-chain alkyl (methyl, ethyl, or propyl) esters obtained from triglycerides by transesterification with alcohol [3,4]. Fatty acids (FAs) are composed of hydrocarbon and carboxylic acid groups, and are responsible for the hydrophobic properties exhibited by lipids. FAs differ in aliphatic chain length, degree of unsaturation, configuration, and position of double bonds, and also have various functions, and most of the commonly found FAs contain even numbers of carbon atoms (C 4 -C 28 ) [5,6]. Algae are widely studied for FAs because of their chemotaxonomic, nutritional, and industrial importance. Lipids are defined as apolar natural compounds, which can be classified by their FA profiles [7]. FA profiles are used as chemotaxonomic markers to define groups of different twigs. Aerophytic algae were first cultivated in 1% Bold's Basal Medium (BBM) with fresh agar. The cultures were maintained under controlled conditions (temperature, 20 • C; 12-h light/12-h dark cycle; 3000 µE m −2 s −1 lx (40-W cool fluorescent tubes). Approximately 2-3 months after the first cultivation of algae, a microscopy study was performed. All phototrophs were observed live and identified using a Jenamed 2 light microscope (Carl Zeiss, Jena, Germany).

Microscopy
Algae were observed and photographed with a Nikon Eclipse Ti inverted microscope, and with Nikon A1 confocal equipment (Nikon, Tokyo, Japan). The 405.488.561-nm laser heads and detection system comprised a PMT-DU4 detector in the range of 400-820 nm, a Plan Apo VC ×100 oil immersion lens, Nikon C-HgFiE mercury illumination, and a DS0Fi1C-U3 digital camera. Digital images were obtained with Nikon NIS-Elements software, and processed with Adobe Photoshop CS5 (Adobe, San Jose, CA, USA).

PCR
Total DNA was extracted from C. subglobosa dry cell mass using the DNeasy Plant Mini kit (Qiagen) according to the manufacturer's instructions. This isolation kit provides silica-based plant DNA extraction in spin column format. For amplification, Coccomyxa-specific oligonucleotide primers were used (F, 5 -ATC CCG ACT TCT GGA AGG GAC GTA-3 ; R, 5 -TCT AGG TGG GAG GGT TTA ACG AA-3 ). The PCR procedure consisted of an initial step for 3 min at 94 • C; followed by 35 cycles of 30 s at 94 • C, 1 min at 56 • C, 1 min 40 s at 72 • C; and a final extension of 5 min at 72 • C [23]. PCR products were subjected to electrophoresis in a 1% agarose gel. The gel was stained with ethidium bromide (0.5 µg/mL) and DNA was visualized under a UV light.

Lipid Extraction and Methyl Transesterification
C. subglobosa cells were cultivated under aerobic conditions in a fermenter (Biostat B, Sartorius Stedim Biotech, Gottingen, Germany) under the same conditions as the algal samples collected from the Głowoniowa Nyża Cave (cultured in the laboratory on Petri plates). Cells were collected (5000 g, 30 min) and lyophilized. Next, the dried cells (5 g) were suspended in 50 mL of a chloromethane-methanol 2:1 mixture for 60 min at room temperature with magnetic stirring. When the extraction was finished, the mixture was filtered with sintered glass in a Buchner funnel, and the solvent was evaporated. After filtration, water was added to create a biphasic system with a final ratio of 8:4:3 chloromethane:methanol:water. The organic phase containing lipids was isolated and vaporized under vacuum. The lipid fraction was suspended in 4.5 mL of a methanol-hydrochloric acid-chloromethane mixture for 4 h at 80 • C with magnetic stirring. The solution was cooled, mixed with 1.5 mL water, and 3 × 4 mL of a hexane-water mixture. The organic phases were collected and filtered with a funnel before GC-MS analysis [24].

GC-MS Analysis of FAMEs
Qualitative and quantitative analyses were conducted by the GC-MS method using the Clarus 600T instrument from PerkinElmer (Waltham, MA, USA) [24][25][26][27]. Compounds were separated on the Elite-5MS capillary column (5% phenyl methyl siloxane; length, 30 m; internal diameter, 0.25 mm; film thickness, 0.25 µm) at 60 • C for 1 min, and then the temperature was increased at a rate of 15 • C per min up to 250 • C, and maintained at 250 • C for 5 min. Total analysis time was 18.67 min. The injection port temperature was 250 • C, and the carrier gas helium was kept at a steady flow rate of 1 mL/min. The ion source and transfer line temperatures were set at 250 • C. The electron ionization energy in spectrometry was 70 eV. FAME identification was carried out with C8-C24 saturated and unsaturated external standards, a GC-grade n-hexane solution, and the NIST Mass Spectra Database.

Results
In this study, we present that the aerophytic alga C. subglobosa isolated from a cave, can be used as a potential source for biodiesel production. Coccomyxa cells were variable in shape, usually cylindrical, elongate-ovoid, or ellipsoid, sometimes asymmetrical, thin-walled, solitary or grouped within a mucilaginous envelope, and with or without concentric striations surrounding cell groups. Irregular, elliptical-to-globular, parietal chloroplasts were plate-like and lateral in position, without pyrenoid or flagellated stages. Each sporangium contained two to four autospores for asexual reproduction. The average cell size was 6-14 × 3-6 µm ( Figure 1).  Different life stages of C. subglobosa were found in all studied samples. Cells were 2-4 µm wide, 3-8 µm long, green-colored (indicating the presence of chlorophyll a and b), and broadly ellipsoid to subspherical to spherical, with mucilage surrounding the cells either homogeneously or with striations, as demonstrated by the Nikon Eclipse Ti inverted microscope with Nikon A1 confocal equipment.  In this work, FAs from C. subglobosa containing C12-C24 were isolated, and FA profiles revealed qualitative and quantitative differences. Figure 3 presents the GC-MS profile of methyl esters of free FAs from C. subglobosa. Additionally, the content, density, and viscosity of FAMEs from C. subglobosa are presented in Table 1. This profile was consistent with the FA profile in seaweed [28]. Lang et al. identified 76 different FAs and ten other lipophilic substances from 2076 FAs profiles of microalgal strains [7]. However, only one genus, Nostoc, belongs to terrestrial habitats (aerophytic algae).  [29]; ** Viscosity measured at 40 °C , estimated according to [30]. In this work, FAs from C. subglobosa containing C12-C24 were isolated, and FA profiles revealed qualitative and quantitative differences. Figure 3 presents the GC-MS profile of methyl esters of free FAs from C. subglobosa. In this work, FAs from C. subglobosa containing C12-C24 were isolated, and FA profiles revealed qualitative and quantitative differences. Figure 3 presents the GC-MS profile of methyl esters of free FAs from C. subglobosa. Additionally, the content, density, and viscosity of FAMEs from C. subglobosa are presented in Table 1. This profile was consistent with the FA profile in seaweed [28]. Lang et al. identified 76 different FAs and ten other lipophilic substances from 2076 FAs profiles of microalgal strains [7]. However, only one genus, Nostoc, belongs to terrestrial habitats (aerophytic algae).  [29]; ** Viscosity measured at 40 °C , estimated according to [30]. Additionally, the content, density, and viscosity of FAMEs from C. subglobosa are presented in Table 1. This profile was consistent with the FA profile in seaweed [28]. Lang et al. identified 76 different FAs and ten other lipophilic substances from 2076 FAs profiles of microalgal strains [7]. However, only one genus, Nostoc, belongs to terrestrial habitats (aerophytic algae). In this study, C12:0, C14:0, C16:0, C18:0, C22:0, and C24:0, and other FAs were identified ( Figure 3). C16 (methyl palmitate) and C18 (methyl stearate) were obtained. Figure 4 presents a portion of the chromatogram with dominant peaks of FAMEs: C16:0 (27.3%), C16:1 (3.1%), C16:2 (0.6%), C18:0 (47.3%), C18:1 (18.9%), and C18:2 (2.8%). Methyl palmitate (C16:0) and methyl stearate (C18:0) were the major saturated FAMEs. Methyl palmitoleate (C16:1) and methyl oleate (C18:1) were the major unsaturated FAMEs.

Discussion
Aerophytic algae offer a potential source (because of their small size and large biomass) for sufficient production of renewable fuels to impact the consumption of fossil fuels. Algae produce large amount of oil from oilseeds and this production is performed by different methods [21]. Furthermore, oil extracted from biomass of algae contains polar lipids, phospholipids and glycolipids, non-polar glycerides and free fatty acids. Marine, freshwater and aerophytic algae species exhibit different concentrations of total saturated and unsaturated fatty acids which a characteristic profile for each. Additionally, the content, density, and viscosity of FAMEs from C. subglobosa are presented in Table 2.  [29]; ** Viscosity measured at 40 • C; estimated according to [30].

Discussion
Aerophytic algae offer a potential source (because of their small size and large biomass) for sufficient production of renewable fuels to impact the consumption of fossil fuels. Algae produce large amount of oil from oilseeds and this production is performed by different methods [21]. Furthermore, oil extracted from biomass of algae contains polar lipids, phospholipids and glycolipids, non-polar glycerides and free fatty acids. Marine, freshwater and aerophytic algae species exhibit different concentrations of total saturated and unsaturated fatty acids which a characteristic profile for each.
The aerophytic alga C. subglobosa isolated from a pseudokarstic cave was selected. Additionally, Coccomyxa simplex (SAG 216-9a), from the Culture Collection of Algae was used in many studies for biofuel and polyunsaturated FA (PUFA) production [31]. Although seaweed has many PUFAs and is a potential source of FAs, the possibility of using unprocessed glycerol, which can be a carbon source for green unicellular algae, such as Chlorella pyrenoidosa and Coccomyxa subellipsoidea [32], is of potential interest. Similar studies considering C. subellipsoidea for medical treatment made use of brewery effluent phytohormones to increase biomass and lipid accumulation in this species [7].
The FA composition of seaweed and aquatic and terrestrial algae differ significantly, but aerophytic algae are especially rich in long chain PUFAs [33]. Table 3 presents chemical compositions of the high lipid content of various algae from different habitats: marine, freshwater, and terrestrial. The genus Coccomyxa was described for the first time by Schmidle, and its classification as a close relative of Dactylococcus in the order Protococcoideae was finally included in the green algal class Trebouxiophyceae, which was separated by molecular approaches [36,37] into the Chlorella, Oocystis and Trebouxia genera [38], mostly consisting of terrestrial species. Darienko et al. stated that all species of Coccomyxa belong to the Elliptochloris clade [39]. The genus Coccomyxa is distributed worldwide in various ecosystems in diverse lifestyles (free-living, parasitic, or as photobionts). This cosmopolitan microscopic algal species forms macroscopic mucilaginous masses in terrestrial and aquatic habitats, rarely free-floating, but sometimes as a lichen phycobiont. Moreover, the Coccomyxa genus occurs as biofilms, in soil, on moss, as planktonic cells in limnetic ecosystems, as symbionts associated with fungi and vascular plants, and as parasites of marine mussels.
However, to date, only a limited number of algal species have been investigated for their FA composition. In this study, we evaluated C. subglobosa aerophytic algae as a potential source of oil products, by determining their total lipid content and FA profile. The total lipid content was different for species from the Coccomyxa genus, because it depends on the environmental conditions in which the algal isolates usually exist or survive. Generally, algae are very capable of surviving, reproducing, and growing in different environmental conditions, and lipid metabolism can be modified in response to habitat changes, which is reflected in the diversity and pattern of cellular lipids [40]. Juneja et al. determined the total lipid content and FA profiles in algae in natural habitats or under the influence of environmental factors and nutrient availability [41]. Jensen showed that lipid content in seaweed is low (from 1% to 5% of dry matter), and is significantly different in various algal species (Table 3) [42]. The aerophytic alga C. subglobosa has higher total lipid content in comparison to the brown alga Padina pavonica and the red alga Jania rubens. Chao et al. investigated C. subellipsoidea as a model alga to augment lipid productivity in oleaginous vascular plants by manipulating nitrogen sources [43]. Seaweed has a low lipid content, and therefore is not very often used for biodiesel production (because of high cost), whereas aerophytic algae are more economical sources of biofuel products. Lang et al. examined 2076 strains from the algal culture collection (Göttingen University); among these were 76 different FAs and ten other lipophilic substances [7]. To define species or the algal taxonomic level, FA distribution patterns can be used as chemotaxonomic markers. Nevertheless, it is difficult to predict the FA profile of a new algal isolate, because of variation at the species level. Considering the diverse FA pattern distribution at the species level, it is difficult to define this profile in a new isolate. FA distribution shows phylogenetic relationships among different taxonomic levels in both genomic and molecular phylogenies.
Darki et al. suggested that amounts and types of fatty acids, along with biochemical composition, vary depending on environmental conditions [44]. Salinity, temperature, pH, and nutrients influence algal growth and biosynthetic lipid production, and promote the formation and accumulation of different lipids, which are indispensable for controlling intracellular stress. Additionally, technical aspects of biodiesel production are crucial, such as at the lipid-extraction stage. For example, ultrasonication is the best method to extract lipids from microalgae [45]. Biodiesel is important sources of renewable, biodegradable and environmentally fuel alternative and produces less harmful gas emissions. Biofuels produce from bio-sources reduce the need for petroleum oil and are favorable for sustainability and reduce pollutant and greenhouse gas emissions e.g., sulphur oxide [18]. Guzman et al. stated that, biodiesel reduces carbon dioxide emissions by 78% in comparison to conventional diesel fuel [27]. Knothe concluded that, for biodiesel production algae with a high proportion of saturated fatty acids are preferred because this leads to higher oxidative stability and higher ignition quality and produces an overall higher quality product [8]. Moreover, the fatty acid methyl ester profile is important such as a factor that determines the suitability of any feedstock using in biodiesel production.
Aerophytic algae synthesize and accumulate high amounts of lipids, proteins and carbohydrates in their cells and therefore they are good potentially sources of biodiesel and bioactive secondary metabolites. Algae can be used as renewable source for a biodiesel production because they are non-competitive and rapidly growing organisms, have capability to accumulate of lipids, metabolic flexibility and a biomass production occur during the whole year. Considering the climate and food problems, also energy crisis in the world, algae are now becoming the main source of biofuel producers. Moreover, algal biofuel does not contain harmful chemicals, so environment can be maintained clean after the combustion. For the clean and safe environment maintaining sustainability, renewable and environmentally friendly fuels are needed to be produce. Our C. subglobosa strain cultured in BBM medium could potentially be applied to biofuel production, by esterification of palmitic acid with methanol, ethanol, and isopropanol.

Conclusions
In this article, we report an aerophytic alga, C. subglobosa, isolated from a cave, which could potentially be used to produce biodiesel. Considering its FA profile, C. subglobosa was rated as a promising oil source. Cell capacity was 8.5 × 10 6 cells/mL culture, corresponding to a dry weight of 5.0 g/mL, producing 32% oil, and FA profiles were identified as C 12 -C 24 . According to chromatographic analysis (GC-MS) the dominant FAMEs were saturated compounds: methyl stearate (C18:0) and methyl palmitate (C16:0) and unsaturated compounds: methyl oleate (C18:1) and methyl palmitoleate (C16:1). Preparing cultures and further developing this algal project could be of great importance in producing biofuel on an industrial scale.