Analysis of growth, substrates utilization and biomass composition

Isolation, screening, identification and characterization of microalgae for neutral lipid accumulation

4.2 Materials and Methods

4.2.4 Analysis of growth, substrates utilization and biomass composition

Analysis of growth, utilization of substrates and biomass composition were carried out at every sampling time point. A known volume of sample was centrifuged at 8000 x g for 10 minutes at 4°C and the supernatant was collected for extracellular substrates analyses (glucose, nitrate and phosphate). The pellet was utilized for the analysis of biomass compositions which includes carbohydrates, proteins, chlorophyll and lipid.

4.2.4.1 Analysis of growth

Cell density was monitored by measuring the absorbance at 690 nm (A690) using a UV-Visible spectrophotometer (Cary 50, Varian, Australia). The protocol for dry cell weight measurements were detailed in section 3.2.6. The absorbance values were converted

in to dry cell weight (DCW) through appropriate calibration equations (Fig. 4.1). For photoautotrophic condition: one cell density = 0.21 g dry cells L^-1 (R² = 0.99); mixotrophic condition: one cell density = 0.27 g dry cells L^-1 (R² = 0.99) and for nutrient starvation: one cell density corresponds to 0.22 g dry cells L^-1 (R² = 0.99).

Fig. 4.1 Correlation graph between the dry cell weight and absorbance measured at 690 nm in a spectrophotometer under (A) photoautotrophic, (B) mixotrophic growth conditions and (C) Nitrogen starved condition

The biomass productivity (𝑃_𝐵, mg L^-1 day^-1) under different cultivation conditions were calculated based on the following equation

𝑃_𝐵 = ^𝑋_𝑡^𝑓−𝑋0

𝑓−𝑡₀ (4.1)

where, 𝑋₀ and 𝑋_𝑓 were the dry cell weight (g L^-1) obtained at initial (𝑡₀) and final (𝑡_𝑓) time points (in days) respectively. Specific growth rate of the cells was calculated based on the following equation

𝜇 =^{𝐿𝑛(𝑋}

2⁄𝑋₁₎

(𝑡₂−𝑡₁₎ (4.2)

where, 𝑋₁ and 𝑋₂ were the dry cell weight (g L^-1) obtained at initial (𝑡₁) and final (𝑡₂) time points (in days) respectively.

4.2.4.2 Analysis of nitrate utilization

Nitrate estimation in the supernatant was carried out using salicylic acid method with sodium nitrate as the standard (Cataldo et al., 1975). In this method, 0.1 mL of the supernatant was mixed with 0.4 mL of 5 % (w/v) salicylic acid in sulfuric acid followed by incubation at 25°C for 20 minutes which yields a yellow colored solution after neutralization with 9.5 mL of 2N NaOH. The absorbance was read at 410 nm after cooling the tubes to room temperature. A correlation standard curve was obtained: One A410 corresponds to 0.93 g L^-1 of nitrate (Fig. 4.2A).

Fig. 4.2 Correlation graph between concentration of the substrates and their respective absorbance in UV-Visible spectrophotometer for estimation of (A) Nitrate; (B) Phosphate and (C) Glucose

4.2.4.3 Analysis of phosphate utilization

Phosphate estimation was carried out using ascorbic acid method with potassium hydrogen phosphate (dibasic) as standard (Parsons et al., 1984). Combined reagent (0.32 mL) comprising (5 N) sulfuric acid, (0.018 M) antimony potassium tartrate, (0.102 M) ammonium molybdate and (0.1 M) ascorbic acid was used for estimating the phosphate content in the supernatant of 2.0 mL. The absorbance was read at 880 nm after incubation for 10 minutes at room temperature and the correlation between phosphate concentration vs

corresponding absorbance is as shown in Fig. 4.2B (One A880 corresponds to 8 mg L^-1 of phosphate).

4.2.4.4 Analysis of glucose utilization

Glucose estimation in the medium was performed using di-nitrosalicylic acid method (Miller, 1959). Supernatant of 3 mL was added to di-nitrosalicylic acid reagent of 3 mL and incubated for 15 minutes in a boiling water bath. The absorbance was read at 575 nm after addition of 1 mL potassium sodium tartrate (40 % w/v) for stabilization. The correlation equation (One A575 corresponds to 1.56 g L^-1 of reducing sugar) is as shown in Fig. 4.2C.

4.2.4.5 Analysis of intracellular carbohydrate formation

Estimation of carbohydrate fraction in the biomass was performed by phenol sulfuric acid method with glucose as standard (Dubois et al., 1956). The pellet obtained after centrifugation was re-suspended in same volume of deionized water and 0.5 mL of the re- suspended pellet was used as the analytical suspension for carbohydrate estimation. Phenol (5 %, w/v) of 0.5 mL was added to 0.5 mL algal suspension, followed by 2.5 mL of concentrated sulfuric acid along the sides of the tube. After equilibration to room temperature for 10 minutes, the contents in tubes were mixed and incubated at 35°C. After 30 minutes, the absorbance was measured at 490 nm (Fig. 4.3A). The correlation curve represents that one A490 corresponds to 0.223 g L^-1 total sugars.

4.2.4.6 Analysis of intracellular protein formation

For protein estimation, cell pellets were subjected to alkaline hydrolysis by boiling with 2N NaOH at 100°C for 15 minutes and then neutralized to pH 7.0 by adding 1.6N hydrochloric acid (Pruvost et al., 2011). The neutralized solution was used for protein estimation using Lowry’s method (Lowry et al., 1951). The correlation curve (One A660

corresponds to 2.32 g L^-1 of protein extracted) was obtained with bovine serum albumin as standard (Fig. 4.3B).

Fig. 4.3 Correlation graph between concentration of the substrates and their respective absorbance in UV-Visible spectrophotometer for estimation of (A) Carbohydrate; and (B) Protein

4.2.4.7 Analysis of intracellular chlorophyll formation

The chlorophyll estimation was carried out using the method provided by Pruvost et al. (2011) which uses 100 % methanol for extraction at 45°C. An absorbance scan of wavelength from 400 to 800 nm was performed and the following equations given by Ritchie (2006) for organisms containing chlorophyll a and chlorophyll b were used for quantification (Eq. 4.3 and 4.4). Total chlorophyll content of the cells was expressed as the sum of chlorophyll a and b.

Chlorophyll 𝑎 (𝑚𝑔 𝐿⁻¹) = (16.52 × [𝐴₆₆₅− 𝐴₇₅₀]) – (8.09 × [𝐴₆₅₂− 𝐴₇₅₀]) (4.3) Chlorophyll 𝑏 (𝑚𝑔 𝐿⁻¹) = (27.44 × [𝐴₆₅₂− 𝐴₇₅₀])– (12.17 × [𝐴₆₆₅− 𝐴₇₅₀]) (4.4) 4.2.4.8 Analysis of intracellular neutral lipid accumulation

For Nile-red based neutral lipid analysis, cell pellet with absorbance 0.7 re- suspended in 1.0 mL of 25 % (v/v) dimethyl sulfoxide was used. Nile red was added to the

re-suspended pellets at the concentration of 4 µg mL^-1 and incubated at 50°C in a water bath for one minute. The fluorescence spectra was obtained in a spectrophotometer (Fluoromax 3, Horiba, USA) with excitation at 480 nm and emission in the region 550 to 650 nm. The auto-fluorescence of algal cells and the intrinsic fluorescence of Nile red were subtracted from the fluorescence of Nile red neutral lipid complex obtained at 580 nm. Triolein (Supelco, USA) was used as standard for Nile-red based neutral lipid estimation and the correlation graph is as shown in Fig. 4.4A.

Fig. 4.4 Standard correlation graph for the estimation of (A) neutral lipid (triolein) by nile- red based assay method in fluorescent spectrophotometer and (B) total lipid as fatty acid methyl esters assayed in gas chromatograph with standard FAME mix C14-C22

The standard was fitted with sigmoidal curve and the equation obtained is as follows with the R² of 0.99:

𝑦 =

^2704.85

1+𝑒[−(𝑥−0.0918) 0.0199⁄ ] (4.5)

Where, y is the fluorescence intensity (in arbitrary units), 𝑥 is the triolein concentration (mg mL^-1) and the non-linearity in the standard curve is due to the instrument sensitvitiy.

Dynamic profile of neutral lipid accumulation in the biomass was obtained by Nile-red based assay method and the total intracellular lipid were measured as fatty acid methyl esters (FAME) along with fatty acid composition in gas chromatography (GC).