Glucose Detecting

Glucose consumption was measured using HPLC (Agilent 1260, Hi-Plex H column). At the beginning, the glucose calibration curve for HPLC was prepared (Appendix 2). Then,

Chlorella vulgaris was cultivated in the presence of glucose. Different samples were taken during microalgae growth till it reached the death phase. The samples were prepared and filtered with 0.2 µm syringe filters to remove any remaining particles and then transferred to HPLC to measure the glucose consumption during microalgae growth.

3.6.3.1

High-performance Liquid Chromatography (HPLC)

High-performance liquid chromatography or HPLC is a chromatographic technique for identifying, quantifying or purifying the individual components of the mixture.

The setup in this study was as follows:

The filtered samples were transferred to the Agilent HPLC system. 10 µl of the prepared sample was injected into the HPLC system, which consisted of an Agilent 1260 Series Liquid Chromatograph, equipped with a quaternary pump (G1311B), auto sampler and column compartment, refractive index detector, and ChemStation software. The separation was done with Hi-Plex H column at a temperature of 65ºC and a flow rate of 0.6 ml/min. The mobile phase used was milliQ water. The total run time under these conditions was 15 min.

Figure ‎3-3: Agilent 1260 HPLC

3.6.4 Lipid Analysis

In this research, two different methods were used for analyzing lipids in Chlorella vulgaris.

3.6.4.1

Folch Method

One of the most common gravimetric techniques is called Folch method, which was invented by Folch and his colleagues in 1953. In this classic method certain weighed samples were homogenized with a chloroform- methanol (2:1) mixture to final dilution 20 folds of the volume. Then the solution was mixed in a vortex and cells were disrupted for 15 minutes in an ultrasonic device (0.5 cycle, amplitude 60%). The samples were kept in ice to avoid heating. In the next step, the mixtures were kept in an Infors HT Shaker- incubator with a speed of 120 RPM and at a temperature of 25ºC overnight. Then the samples were filtered and the extracted liquid was mixed with 20% water (v/v), centrifuged for 15 minutes at 4ºC and 3500 RPM. In the last step, the lighter phase was removed, the solvent was evaporated and the lipid was recovered and weighed (Floch et al., 1956).

3.6.4.2

Nile-red Staining Method

In order to determine the amount of neutral lipid in the samples a 96-well plate was used. For this purpose, 100 μl of aliquot sample was pipetted into a Corning 3603 black-sided clear bottomed plate and 100 μl of the Nile Red dye (1 μg/ml) solution, prepared in 50% DMSO, was added. Nile red (9-(Diethylamino)-5H benzo [∞] phenoxa- zin- 5-one) is a red phenoxazone and lipid soluble dye which can be used to detect neutral lipids in vivo. Although this dye is very poorly fluorescent in aqueous solutions but it is quite photo- stable and highly fluorescent in non-polar hydrophobic environments. Nile red is dissolved in Dimethyl sulfoxide (DMSO) so that it can pass through the thick and rigid cell walls in many green algae. To read the fluorescence intensity the plate was kept in the Tecan plate reader for 30 minutes. Fluorescence intensities were measured from the top using 530 nm excitation and 570 nm emission wavelengths with a Tecan M1000 plate reader and the readings were recorded (W. Chen et al., 2011, 2009; Held & Raymond, 2011). Nile red was purchased from Sigma- Aldrich.

3.6.4.3

Correlating Fluorescence Data with Gravimetric

Measurements

Folch method is a classic gravimetric method that can be used for calculating the total lipid content of microalgae. Unfortunately, this method is time consuming and this is one of the weaknesses of the technique (Bligh & Dyer, 1959b). On the other hand, the amount of neutral lipids can be calculated with Nile-red method which uses Nile-red dye as a lipophilic stain. The fluorescence intensity corresponding to the sample and dye could be quantified with Tecan M1000 plate reader (Held & Raymond, 2011). One of the goals in this study was to find a fast and reliable technique for calculating the total lipid content by measuring the fluorescence intensity.

In the first step, a 4-liter medium with a concentration of 15 g/L glucose was prepared in a bioreactor. Chlorella vulgaris was cultivated inside the bioreactor. The cultivation process continued until the microalgae reached the stationary phase which contains the highest amount of lipid. 20 ml of culture was used for preparing twofold dilution series. Absorbance was measured and the neutral lipid was identified with Nile-red staining

method. The rest of the culture was centrifuged at 4ºC with a speed of 3500 RPM for 20 minutes. The residue was washed 3 times with distilled water in order to remove the remaining salt of the medium from algae biomass. The wet biomass was transferred to the freezing dryer for dehydration and drying. In the next step, the total lipid content of lyophilized algae powder was calculated using the Folch method.

With the purpose of calculating the total lipid from neutral lipids by measuring the fluorescence intensity of microalgae samples, the obtained data from Folch and Nile-red method were correlated. The curve in Figure 3-4 was used as the calibration curve to estimate the lipid content in an algae culture using the fluorescence intensity.

Figure ‎3-4: Amount of total lipid content in algae versus fluorescence intensity from Nile-red method (±SD).