Molecular identification

The 18S rRNA gene and ITS sequences obtained for strain HSO-3-1 were submitted to the National Center for Biotechnology Information (NCBI) under accession numbers JF930340 and JF930341, respectively. The amplified 18S rRNA gene sequence was found to have > 99% identity with that of previously sequenced Mychonastes sp. and Pseudodictyosphaerium sp. strains in the NCBI database. The 18S rRNA gene analyses of 26 strains belonging to the genus Mychonastes clade allowed the recognition of lineages attributable to 10 different species (Figure 1). On the 18S rRNA gene tree, HSO-3-1 was found to be most similar to the M. afer strain CCAP 260/6 (GQ477044.1). To confirm the results of the 18S rRNA gene analyses, the ITS1-5.8S-ITS2 sequence of 26 strains belonging to the genus Mychonastes clade was studied, and allowed the recognition of lineages attributable to eight different species (Figure 2). Strain HSO-3-1 was again found to have a close relationship with M. afer strain CCAP 260/6 (GQ477044.1) using this analysis.

Morphology and cellular structure

Cells of M. afer HSO-3-1 are unicellular, solitary and spheroidal, and the cell size is very homogeneous, varying from 2 to 3 μm in diameter. The cell wall is smooth, and has a layer of along the central axis when examined under scanning electron microscopy (Figure 3, left). The cell wall consists of a double layer (Figure 3, right). The outer layer, which is 50 to 200 nm wide, was found to be a trilaminar sheath, consistent with a previous report [17]. Each cell has a single chloroplast that is crescent-shaped and anchorage-dependent (Figure 4A-D). The chloroplast consists of thylakoid lamellae arranged in six almost parallel rows with pyrenoids (Figure 4A-D). Reproduction takes place by the formation of two autospores (Figure 4E-F). Lipid bodies are often present in the cytoplast after cultivation for a significant period (Figure 4G-H).

Cells of strain HSO-3-1 are single (seldom found in colonies) and planktonic. The adult cells are spherical, 2 to 5 μm in diameter, with a thin mucilaginous envelope. The shape of HSO-3-1 was found to be similar to that reported for M. afer [18].

Morphological features of strain HSO-3-1, including the shape of the cells, the composition of the cell wall, and the shape and composition of the chloroplast, are identical to those reported for the genus Mychonastes [18] and the species Mychonastes homosphaera [19]. However, the autospore formation of HSO-3-1 involves binary fission, and is different to that of M. h20omosphaera. The size of the cells is more homogeneous, and the cell wall is thicker than M. homosphaera. In contrast to the genus Pseudodictyosphaerium, which is characterized as having a parietal, cup-shaped or girdle-shaped chloroplast without pyrenoids, the chloroplast of HSO-3-1 consists of thylakoid lamellae with pyrenoids. Based on these molecular and morphology characteristics, strain HSO-3-1 was therefore identified as M. afer.

Fatty acid identification

The major FA component was C18:1, which accounted for more than half of the total FAME content in M. afer HSO-3-1. Biodiesel quality is determined by FA composition [17]. It has been suggested that high-quality biodiesel is usually composed of 18 carbon acids, including oleic acid methyl ester and octadecenoic acid methyl ester [20]. The major FAMEs contained in M. afer HSO-3-1 were shown to be hexadecanoic acid methyl ester, octadecenoic acid methyl ester and octadecenoic acid methyl ester, which accounted for over 57.6% of the total lipid content.

The second major FA was C16:0. The analysis also revealed the presence of C24:1 (nervonic acid, 14.7 mg/g of total lipid, 3.8% of total FAs), which has been shown to have biomedical applications [21, 22]. The production of nervonic acid by M. afer HSO-3-1 increases the value of this microalgal strain for biodiesel production. Nervonic acid is a long-chain monounsaturated FA, which is found in the seed oils of Lunaria annua (honesty), Borago officinalis (borage), hemp, Acer truncatum (Purpleblow maple) and Tropaeolum speciosum (Flame flower). Only Lunaria annua L. has been studied and grown sparingly for future development as a niche crop [21, 22]. Therefore, the discovery of C24:1 in microalgae expands the variety of natural sources of C24:1, and thus improves the potential value of this microalgal strain in biodiesel production, as it can also be used for pharmaceutical production [17].

Effect of NaNO₃concentration on lipid accumulation

The effect of nitrogen concentration on the growth and lipid accumulation of M. afer HSO-3-1 was investigated. HSO-3-1 was grown at different NaNO₃ concentrations. When the concentration of NaNO₃ was in the range 0.15 to 3.15 g/l, the lipid content of the algae was between 41.7 and 51.9% DCW after cultivation for 14 days, with the highest biomass (3.29 g/l) and lipid yield (1.62 g/l) achieved when the concentration of NaNO₃ was 3.15 g/l (Figure 5). The highest lipid content (51.9% DCW) was achieved at a NaNO₃ concentration of 1.65 g/l.

The total content of lipids in microalgae may vary from about 1 to 85% of dry weight [21, 23]. Hu et al. summarized the lipid contents of oleaginous green algae reported in the literature, and found that oleaginous green algae had an average total lipid content of 25.5% DCW [17, 24]. Nitrogen limitation/depletion has been shown to affect both lipid composition and lipid content of many algae [25]. The promotion of FA synthesis seen during the decay phase was probably due to triglyceride-FA accumulation [15, 26]. Rodolfi reported that the lipid content of Nannochloropsis sp. increased from 32% to 60% when it was transferred from 'nitrogen-sufficient' to 'nitrogen-deficient' culture conditions. Usually microalgae accumulate lipids under nitrogen-limiting conditions, when energy and carbon sources are available [27]. However, limitation or depletion of nitrogen did not seem to induce lipid accumulation in strain HSO-3-1. Interestingly, using transmission electron microscopy (TEM), we found that the cell wall became thicker when the size and number of lipid bodies dramatically increased in M. afer HSO-3-1 (Figure 3, Figure 6). It is unclear whether there is any correlation between the cell-wall structure and lipid body formulation. Thus, the metabolism of lipid accumulation in M. afer HSO-3-1 under different nutrient conditions should be investigated further. The lipid content of the new species, M. afer HSO-3-1 was up to 51.9% DCW when the alga was grown in BG-11 medium, indicating that the strain could be used as a suitable feedstock for biodiesel production.

Organism and culture conditions

Strain HSO-3-1 was isolated from wastewater samples collected from Jinan, Shandong province, and stored in our laboratory. The strain was grown in BG-11 medium [28], and maintained at 25 ± 1°C under continuous illumination provided by daylight fluorescent tubes at 70 to 80 μmol photosynthetically active radiation (PAR) photons per m² per second.

Electron microscopy

For TEM, cells were fixed with 1% glutaric dialdehyde for 1 hour, separated by centrifugation for 5 minutes at 903×g, then washed three times with phosphate buffer (pH 7.2 to 7.4). The processed materials were fixed with 1% chenic acid for 1 hour and then washed three times with phosphate buffer (pH 7.2 to 7.4). The cells were dehydrated with increasing concentrations of ethanol up to 100% and anhydrous acetone. They were then soaked in an anhydrous acetone:Epon812 resin (7:3) mixture for 5 hours, followed by an epoxy propane:Epon812 resin (3:7) mixture for 6 hours, then Epon812 for 5 hours, after which they were finally embedded in Epon812 resin. Photographs were taken under the microscope (H-7650; Hitachi, Tokyo, Japan). All experiments were carried out at room temperature.

For scanning electron microscopy, the cells fixed with glutaric dialdehyde were embedded using the GTGO (glutaraldehyde, tannic acid, guanidine hydrochloride, osmium tetroxide) technique [29], coated with gold, and examined under a microscope (S-4800; Hitachi, Japan).

Genomic DNA preparation

Induction of lipid production at different NaNO₃concentrations

Microalgae were cultivated in Erlenmeyer flasks to the exponential phase of growth, then transferred to 500 ml glass bubble columns (41 mm in diameter) containing 250 ml of BG-11 medium with different concentrations of NaNO₃ at a 1:6 (v/v) dilution, to maintain the OD₇₅₀ value at around 0.2. The nitrogen concentration was monitored during growth by measuring OD₂₂₀ and OD₂₇₅ after adding 0.02 mol/l HCl and 1.65 × 10^-4 mol/l sulfamic acid according to Feng's method [33, 34].

The columns were maintained at 25 ± 1°C, and bubbled with sterile gas composed of air supplemented with 2% (V/V) CO₂. Continuous artificial illumination at 270 ± 20 μmol PAR photons/m²/s¹ was provided by daylight fluorescent tubes on one side. All experiments were performed in duplicate.

Growth was estimated by measuring the dry cell weight (DCW). A 5 ml sample was taken every 12 hours and filtered through pre-weighed 0.8 μm microporous filter paper. The filter paper was oven-dried overnight at 105°C. The difference between the final weight and the weight of the paper before filtration was taken as the DCW.

Lipid extraction

The microalgal cells were harvested after 13 days by centrifugation at 4722×g for 10 min. Cell pellets were lyophilized using a freeze drier (Alpha1-2LD Plus; Martin Christ GmBH, Osterode, Germany). The total lipids contained in the algal cells were extracted with a modified chloroform-methanol-water solvent system [35]. In a 10 ml tube (tube 1), 30to 40 mg dry algal powder, 4 ml chloroform and 2 ml methanol were mixed, and shaken for 10 seconds to disperse the powder. The tube was then incubated for 12 hours at 30°C while shaking at 200 r/min and separated by centrifugation for 10 minutes at 903×g, then 6 ml of the supernatant was collected and transferred to tube 2. Into this, 2 ml methanol and 3.6 ml deionized water were added to give a final chloroform:methanol:water ratio of 10:10:9. The contents were separated by centrifugation at 903×g for 5 minutes, then the chloroform layer was removed, transferred into a pre-weighed tube (tube 3; weight 1) and dried for 30 min under a flow of N₂ at 60°C. Tube 3 was dried under vacuum (Lantian DZF-6050, Hangzhou, China) for 1.5 hours at 60°C, and then weighed (weight 2). The lipid weight was calculated by subtracting weight 1 from weight 2.

Fatty acid analysis

Methyl esters were generated from the microalgae lipids by heating them in a 2% H₂SO₄-methanol solution at 85°C for 2.5 hours. Analysis of the FAMEs was then performed using GC-MS (7890A-5975C, Agilent Technologies Inc., Wilmington, DE, USA) with a fused column (30 m × 0.25 mm; HP-INNOWAX; Agilent Technologies Inc.). The carrier gas was helium, and 1 μl of the methyl ester sample solution was injected for each analysis. The split ratio was 1:20. The temperature program was as follows: the column temperature was maintained at 50°C for 1 minute, increased to 200°C at a rate of 25°C/min, then increased to 240°C at a rate of 3°C/min, and finally maintained at this temperature for a further 15 minutes. The injector temperature was set at 260°C. The identification of FAs was performed by comparing the mass spectra obtained with Wiley libraries. http://www.wiley.com/WileyCDA/WileyTitle/productCd-1118143949.html