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Appl. Sci. 2020, 10, 2531 3 of 18 decreased by 10 μE m−2 s−1 using the red sheet. The growth of microalgae was measured by optical density at 680 nm every 48 h in control white light. The growth curve represented the number of cells plotted as a function of time and could be divided into four phases according to the slope representing the four stages lag, exponential, stationary, and death. 2.2. Experimental Setup for Autotrophic and Mixotrophic Culture under Different Light Colors The study was divided into two series of experiments. The first set of preliminary experiments studied the impact of three light colors: Red (R), Yellow (Y), and White (W) in both mixotrophic and autotrophic conditions under the growth conditions explained above. All the experiments were conducted into 250 mL Erlenmeyer flasks containing 50 mL of liquid media with and an initial inoculum size of 0.2 OD. For mixotrophic cultivation, glucose or glycine was added to the L1 media at a concentration of 1%, which were labeled as 1 and 2, respectively. The experiment identifications are shown in Table 1. In this experiment, all the cultures were exposed to the conditions for the complete culture duration (i.e., from day 1 to day 10). We harvested the cultures at day 10 during the stationary phase. Table 1. Summary of P. tricornutum culture conditions and experiment identifications. P. tricornutum was cultured in L1 media, and all experiments were done in triplicate. Light Autotrophic Mixotrophic 1% Glucose 1% Glycine White (Control) W W1 W2 Red R R1 R2 Yellow Y Y1 Y2 In the second series of experiments, the microalgae culture in autotrophic growth conditions was exposed to a light shift from red light to white light in different growth phases (Table 2). For this, P. tricornutum culture was submitted to red light treatment during the specific growth phase i.e., lag, exponential, stationary, and then cultures were exposed to white light labeled as RT1, RT2, RT3, and RS (Table 2). All experiments were done in triplicate. All the inoculum for these experiments were grown in control white light. Table 2. Overall experimental plan of four culture conditions (red light treatment 1 to 3 and RS). After the R exposure using cellophane sheets during the treatment time period as mentioned for (RT1–RT3, RS), the sheets were removed, and P. tricornutum culture was exposed to the full spectrum of white light. In RT1, the culture was exposed to red light for first 4 days and then exposed to white light. In RT2, the culture was exposed to white light during first 3 days; then, it was exposed to red light by wrapping the flask from the fourth to seventh day and then again exposing the flask to white light. In RT3, the culture was exposed to white light until the seventh day and after was given red light treatment. Similarly, in RS, the red light was given from the first to seventh day and then exposed to white light for 72 h before harvesting. Below, the graphical representation of the setup aligned with the growth curve. RT1 RT2 RT3 RS Description Red light exposure only during the lag phase (1st–4th day) Red light exposure only during the exponential phase (4th–7th day) Red light exposure only during the stationary phase (7th–10th day) Appl. Sci. 2020, 10, x FORRPEeEdR lRiEgVhIEtWuntil the end of the exponential phase (1st–7th day) 4 of 22 . 2.3. Cell Growth and Biomass Analysis Wild-type P. tricornutum was cultured in 5 mL of L1 medium in a flat-bottomed sterile 6-well culture plate with a lid (Costar® , Cat no. 3516). The cultures were maintained with consistent shaking on an orbital shaker at 130 rpm. Cell growth was monitored by measuring the optical density at 680 nm using a Synergy H1 Microplate Reader, BioTek. All the conditions were studied in triplicate. The biomass was estimated by dry weight from the centrifuging of P. tricornutum cultures at 7000× g for 10 min on the 10th day. The supernatant was discarded, and the pellet was dried at 60 °C for biomass analysis until the constant mass was achieved. Treatment 2.4. Visualization of Lipid BodiesPDF Image | Red Light Variation Lipid Profiles in Phaeodactylum tricornutum
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