Cyanobacterial cultivation
The unicellular halotolerant cyanobacterium A. halophytica was maintained by cultivation on BG11 agar (pH 7.4) (Rippka et al. 1979) supplemented with Turk Island salt solution (Garlick et al. 1977) at 30°C under a white-light illumination of 30 µmol photons m− 2 s− 1. In this experiment, axenic cells of A. halophytica were grown in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with various NaNO3 concentrations (0.176–17.6 mM). BG11 contains NaNO3 at a final concentration of 17.6 mM. An initial cell concentration of cyanobacterial culture was adjusted to the optical density of about 0.1 at 730 nm. The culture was shaken at 120 rpm under a white-light intensity of 30 µmol photons m− 2 s− 1 (for 18 h day− 1) at 30°C for 14 days. Natural seawater used in this study was collected from Nang Rum Beach, Chonburi Province, Gulf of Thailand, Thailand (12° 36.969’ N, 100° 55.280’E). The seawater was filtered through a glass microfiber filter GF/F (0.7µm) (Whatman, UK) and adjusted to pH 7.5 with 2 N NaOH prior to sterilization by autoclaving. A. halophytica cultured in BG11 supplemented with Turk Island salt solution was used as a control in all experiments.
Growth determination by chlorophyll a content and total cell concentration measurements
Growth of A. halophytica was determined by measuring chlorophyll a content and total cell concentration. For chlorophyll a concentration measurement, cells were collected by centrifugation of one mL of cell culture at 7,000×g at 4 0C for 10 min. Chlorophyll a was extracted from the cell pellet by adding 1 mL of 90% (v/v) methanol and subsequently incubating at room temperature under darkness for 1 h. The chlorophyll a concentration of the extract was determined by measuring the absorbance at 665 nm and calculated according to Mackinney (1941). Cell concentration was determined based on the direct cell count method using Neubauer hemocytometer (Boeco, Germany) under a light microscope (Olympus CH30RF200, Japan). The specific growth rate (µ) was calculated according to Eq. 1 (Tang et al. 2011).
Specific growth rate (µ) (day− 1) \(=\frac{\text{ln}\left({x}_{1}\right)-\text{ln}\left({x}_{0}\right)}{{t}_{1}-{t}_{0}}\) ……..Eq. 1
x1 and x0 are cell numbers at the end and the beginning of the exponential phase, respectively, whereas t1 and t0 are durations at the end and the beginning of the exponential phase, respectively. The doubling time required for the population cells to double its cell number was calculated according to Eq. 2 (Guillard 1973).
Doubling time (day) = \(\frac{\text{ln}2}{\mu }\) ……..Eq. 2
Effect of nitrogen concentration and trace elements in culture medium on growth
A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater containing 0, 0.176, 1.76 and 17.6 mM NaNO3. Cells were shaken at 120 rpm under a white-light intensity of 30 µmol photons m− 2 s− 1 (for 18 h day− 1) at 30°C for 14 days. Chlorophyll a concentration and total cell concentration were measured every 2 days. To increase growth of A. halophytica, Turk Island salt solution containing 0.5 M NaCl, 49 mM MgCl2.6H2O, 30 mM MgSO4.7H2O and 8.9 mM KCl (Garlick et al. 1977) was added in natural seawater media.
Total lipid extraction
A. halophytica cells cultivated in various types of media for 14 days were harvested by centrifugation at 7,000×g at 4°C for 10 min. Cells were dried in a hot air oven at 60°C for 24 h. Lipid extraction from dried cyanobacterial cells was performed by the single-step lipid extraction method (Axelsson and Gentili 2014). Briefly, 20–30 mg of A. halophytica dry cells were added with 8 mL of chloroform and methanol (2:1, v/v). The mixture was shaken vigorously. Two mL of 0.73% (w/v) NaCl was added in the mixture. The sample was mixed and centrifuged at 7,000×g at room temperature for 5 min. The chloroform layer at the bottom of the tube containing the crude lipid extract was then separated and put into a new tube. Lipid extraction was performed repeatedly five times. The chloroform layer in the lipid crude extract was collected and evaporated using a vacuum evaporator and the crude lipid extract was then weighed. The lipid content was calculated according to Sivaramakrishnan and Incharoensakdi (2017) and expressed as percentage of lipid per dry cell weight. Lipid productivity was expressed as a unit of extracted lipid weight per culture volume per day. Dry biomass of cyanobacterial cells was obtained from a centrifugation of cell culture at 7,000×g at 4°C for 10 min before drying the cell pellet in a hot air oven at 60°C for 48 h.
Effects of carbon source and concentration on lipid content and lipid productivity
A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO3 and Turk Island salt solution. Various carbon sources, i.e., glucose, fructose, sucrose, and lactose, were separately added in the medium at a final concentration of 0.189 mmol C-atom L− 1 which equals to Na2CO3 concentration in BG11. A. halophytica cells were cultivated under previously described conditions for 14 days. Cells were subsequently harvested by centrifugation and total crude lipid was extracted by the previously described protocol. To investigate the optimal concentration of the selected carbon source, different concentrations of the selected carbon source were varied at 0.189, 1.89, 18.9, 189, 379 and 756 mmol C-atom L− 1.
Effects of NaCl concentration on lipid content and lipid productivity
A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO3 and Turk Island salt solution. NaCl was added to the medium to reach the final external concentrations at 0, 0.25, 0.5, 0.75, 1, 2, and 3 M. Cells were grown under previously described conditions. After 14 days of cultivation, cells were harvested by centrifugation and total crude lipid was extracted.
Effects of initial pH and temperature on lipid content and lipid productivity
A. halophytica was cultivated in a 250-mL Erlenmeyer flask containing 100 mL of natural seawater supplemented with 17.6 mM NaNO3, Turk Island salt solution and 0.75 M NaCl. To investigate the effect of initial pH on lipid production, pH of the medium was adjusted to 6, 6.5, 7, 7.5, 8 and 8.5, using HCl and NaOH. Cultivation temperature was tested at 20, 25, 30, 35, and 40°C. After 14 days of cultivation, cells were harvested by centrifugation and total crude lipid was extracted, as described above.
Biodiesel properties of fatty acid methyl ester
Several biodiesel properties, i.e., percentage content of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA), degree of unsaturation (DU), saponification value (SV), iodine value (IV), cetane number (CN), long chain saturated factor (LCSF), cold filter plugging point (CFPP), cloud point (CP), pour point (PP), allylic position equivalent (APE), bis-allylic position equivalent (BAPE), oxidation stability (OS), higher heating value (HHV), kinematic viscosity (υ), and density (ρ) were determined based on the fatty acid composition of the obtained methyl ester, by the Biodiesel analyzer software ver. 2.2 (Talebi et al. 2014).