Cell culture and heat treatment
Unicellular chlorophyte Scenedesmus vacuolatus SAG 211-8b was cultured photoautotrophically in semi-continuous mode in a medium similar to that of Chen and Lorenzen (1986). Axenic cultures (100 mL) were grown at 32°C with slow bubbling at 3.5% CO2 in air. Illumination was provided by daylight fluorescence tubes at an intensity of 150 μmol photon m-2 s-1. Synchronous cultures were obtained by the programmed 14-h light/10-h dark regime. The culture started with a cell density of 2 × 106 cells mL-1, and dilution (~15-16×) was made after each 24-h cycle, at which time cell density reached about 3 × 107 cells mL-1. The cell number was determined using a hemacytometer under a light microscope.
When the culture was in the small vegetative cell stage, it was divided into two aliquots. Heat treatment was applied by incubating one 50-mL aliquot of small vegetative cells at 46.5°C for 1 h in the dark. The treated cells were then recultured under the same conditions as before the heat treatment, but in the dark. Another 50-mL aliquot of cells, which served as the control, was cultured in the dark directly without heat treatment. The dark cultivation lasted up to 105 h, during which samples of heat-treated and untreated cells were respectively taken for analysis at various time points as indicated below. Each experiment consisted of three independent measurements with two replicates each.
Cell proliferation ability
The ability of cell proliferation was accessed by colony formation assay. A fixed number of cells (~ 4000 cells) was collected by withdrawing an aliquot of cell suspension at the time points of 0, 6, 8, 12 h and thereafter, at an interval of 12 h up to 84 h of dark cultivation. Samples were streaked onto agar plates containing 1.5% agar and the culture medium. The loaded plates were kept under the conditions similar to that of suspension culture (14-h light/10-h dark regime at 150 μmol photon m-2 s-1 and 32°C), but without CO2 supply.
The numbers of green colonies were counted 14 days later. Each plate (8.4 cm in diameter) was first photographed under a stereomicroscope, five circular areas with a diameter of 1 cm were then randomly selected and colonies in each circle were counted on a computer screen with a magnification of 10×. The number of green colonies on a plate was calculated by multiplying the average count of the five circles by the area ratio (70.56).
Photosynthetic activity
A pulse amplitude modulated fluorometer (PAM 2000, Walz, Germany) was used to measure the in vivo chlorophyll fluorescence of Scenedesmus cells. The minimal chlorophyll fluorescence (Fo) was measured on dark-adapted samples with excitation at 655 nm (0.5 μmol photon m-2 s-1) and detection longer than 700 nm. The maximal fluorescence (Fm) was determined by application of a saturating pulse (< 710 nm) at 2000 μmol photon m-2 s-1 for 0.8 s. The maximum quantum yield (Fv/Fm) was calculated using the equation Fv/Fm = (Fm – Fo)/Fm.
During the heat treatment, the activity was measured at the time points of 0, 0.5, 1, 2, 4, 6, 8, 10, 15 and 20 min. During the dark cultivation, the activities of both heat-treated and untreated cells were measured at the time points of 0, 2, 6, 12 h and thereafter, at an interval of 12 h up to 84 h of dark cultivation. In addition, light (150 μmol photon m-2 s-1) was turned on during the dark cultivation of heat-treated cells at 12, 24, 36, 48, 60 and 72 h, respectively, and the Fv/Fm ratio was closely monitored at an interval of roughly 3–6 h.
Chlorophyll content
Heat-treated or untreated cells (2 × 107) were sequentially removed from the culture after cultivation in the dark for 0, 6, 12, 24, 48, 60, 72 and 85 h, centrifuged at 1,800 × g for 5 min and resuspended in 1 mL of 100% methanol. The cell suspensions were then incubated at 60°C for 30 min in the dark, followed by cooling at 0°C for 5 min, and centrifugation at 1,800 × g for 10 min. Chlorophyll concentration of the supernatant was determined photometrically by using the following equation: Chlorophyll concentration (mg L-1) = 25.5 × A650 + 4 × A665, where A650 and A665 are absorbance at 650 and 665 nm, respectively (Holden, 1976).
To further check the uniformity of chlorophyll content within a cell population, flow cytometry analysis was carried out with a FACSCalibur™ flow cytometer (Becton Dickinson, California). Cell samples (2 mL) of untreated cells at the beginning of dark cultivation and heat-treated cells that had been cultivated in the dark for 0, 48 and 72 h were respectively collected, and the intrinsic fluorescence of chlorophyll a was detected with excitation at 488 nm and emission longer than 650 nm. 104 particles were analyzed in each measurement.
Succinate dehydrogenase activity (MTT assay)
The succinate dehydrogenase activity assay of mitochondria was based on Ikegawa et al. (1998) with some modifications. Scenedesmus cells (2 × 107) were collected by centrifugation at 1,800 × g for 5 min, resuspended and incubated in 1 mL phosphate buffered saline (PBS) containing 1 mg mL-1 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) in the dark at 32°C for 2 h. To quantify the amount of formazan precipitate formed in a cell population, the stained cells, after PBS washes, were resuspended and incubated in 1 mL acid-isopropanol (0.04 N HCl in isopropanol) for 5 min to dissolve formazan crystals. Samples were then measured for absorbance at 590 nm. The MTT assay was done on both heat-treated and untreated cells at the time points of 0, 12, 24, 48, 78, 96 and 105 h of dark cultivation.
Morphological studies
TEM was conducted following the procedure described by Chen and Lai (1996). Scenedesmus cells (6 × 107) were fixed 4 h in 2% glutaraldehyde in 66mM potassium phosphate buffer (pH=7.1) at 4°C, and then post-fixed in 2% aqueous osmium tetroxide in 66 mM potassium phosphate buffer (pH=7.1) for 4 h at 4°C. This was followed by three washes at 15 min intervals with the same buffer. Fixed cells were dehydrated in a grade acetone series, then embedded in pure Spurr resin and polymerized at 70°C for 15 h. Ultrathin gold sections were cut on a Reichert-Jung ultramicrotome, collected on formvar-coated grids, stained with saturated solution of uranyl acetate in 100% methanol, and post-stained with lead citrate. Observations were made using a Hitach-7500 transmission electron microscope (Hitachi, Japan).
Evens blue staining
Scenedesmus cells were incubated with Evans blue (2.5 mg mL-1) in 66 mM potassium phosphate buffer (pH=7.1) for 1 h at room temperature. Unbound dye was removed by three washes with the same buffer. Cell counting was carried out using a hemacytometer under a light microscope. The counting was done on both heat-treated and untreated cells at an interval of 12 h up to 108 h of dark cultivation.