Influence of crude glycerol on the biomass and lipid content of microalgae

Optical panel photobioreactor (OPPBR) construction and operation

A schematic diagram of the OPPBR is shown in Figure 1(a). The three OPPBRs were operated in parallel at a 15 L working volume and each was equipped with an optical panel (OP). The initial concentrations of the inoculated microalgae were 0.357 g/L ± 0.7 g/L of C. vulgaris, 0.342 g/L ± 0.7 g/L of B. braunii and 0.367 g/L ± 0.6 g/L of Scenedesmus sp. The experiments were conducted at neutral pH (7.2 ± 0.3) under dark and light cycles of 8 and 16 h, respectively. The temperature was maintained at 23 °C ± 1 °C using LEDs for 20 days. The OPPBRs were aerated continuously at an aeration rate of 0.5 L/min. CO₂ at the equivalent aeration rate of 0.02 vvm was used for cultivation. CO₂ with a flow rate of 0.74 L/min was introduced into the reactors. The OPPBR was designed such that the light source (22 LEDs), an LED panel (bar type), was placed in the OPPBR. A v-grooved OP was inserted underneath this in the photobioreactor (PBR). The thickness of the OP was 6 mm (Figure 1(b)).[Citation16] For greater functionality, the incident light was uniformly distributed across both sides of the OP in the reactor. A LED light source was used because it is efficient and provides the required wavelength light from 430 nm to 670 nm, which is selective for microalgal growth. Moreover, the light intensity representing the amount of light used for photosynthesis was 200–250 µmol/(m² s). The mixotrophic conditions for algae cultivation were achieved with crude glycerol purchased as a by-product of biodiesel production. The corresponding amount of crude glycerol (0, 2, 5 and 10 g/L) was added to the JM growth medium to prepare the desired mixotrophic medium.

Figure 1. A schematic diagram of OPPBR (a). Optical panel with LEDs (b). Reprinted with permission from Springer: [Citation16] [Choi HJ, Lee SM. Effect of optical panel thickness for nutrient removal and cultivation of microalgae in the photobioreactor. Bioprocess Biosyst Eng. 2014;34:697–705.

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Optical panel in photobioreactor

The characteristics of the OP are listed in Table 1.[Citation16] The OP exhibited 93% transmittance and 1.19 specific gravity. The OP dimensions were 210 mm (L) × 290 mm (H) × 6 mm (W) as shown in Figure 1(b) and were constructed from a transparent panel of pure PMMA (poly-methylmethacrylate) sheets. This material has good transparency and its light absorption in the visible region is negligible.[Citation17] In this study, a v-cut OP (Figure 1(b)) was introduced to evaluate and assess the quantitative effects of crude glycerol on cell growth, biomass productivity and lipid content. With the v-cut technology, the light was guided into the v-grooves that have x-, y- and z-direction dimensions, such as enlarged horizontal and vertical grooves. The vertical v-grooves are widely spaced when they are close to the light source and narrow when distant from the light source. The enlarged horizontal v-grooves are arranged in straight lines along the x-direction from the end edge of the OP and have maximum enlarged portions located on the other edge of the OP. [Citation16] In addition, the v-cut provides a uniform distribution of light in the PBR.

Measurement of cell weight and specific growth rate

The effect of crude glycerol concentrations (0, 2, 5 and 10 g/L) added during the initial growth phase was evaluated in relation to the growth of the algal biomass and lipid accumulation.

To determine the biomass concentration, a sample of microalgae in growth medium was centrifuged for 10 min at 628 g, washed with distilled water and then dried in an oven at 105 °C for 24 h to constant weight. The biomass productivity P (g/(L·day)) was calculated from the variation in biomass concentration X (g/L) within a cultivation time t (in days), according to the following equation: (1) $$P=\left(X_1-X_0\right)/\left(t_1-t_0\right)$$(1)

The specific growth rate µ (in days) was calculated using EquationEquation (2)(2) $$\mu =\ln \left(X_1/X_0\right)/\left(t_1-t_0\right)$$(2) : (2) $$\mu =\ln \left(X_1/X_0\right)/\left(t_1-t_0\right)$$(2) where X₁ and X₀ are the biomass concentration (g/L) on days t₁ and t₀, respectively.

Extraction of lipids

The algal biomass for lipid extraction was prepared by centrifugation and drying. After oven drying, the algae were pulverized and subjected to Soxhlet extraction. All Soxhlet extractions were performed for 72 h using 500 mL of solvent per 1 g of pulverized dry algae with a cycle time of 10–15 min. Soxhlet extraction with hexane was used because the Bligh and Dyer [Citation18] extraction method is suitable for extraction of all lipids, including triglycerides, phospholipids and other pigments.[Citation19] The lipid content does not reflect the exact amount of triacylglycerols (TAGs; consisting of a glycerol moiety with each hydroxyl group esterified to a fatty acid) because only triglycerides are used in the synthesis of biodiesel and other components are undesirable. The excess hexane was evaporated by rotary evaporation until the total volume reached 30– 40 mL. The solutions were diluted to 50 mL and used to determine the TAG content. The amount of TAG was determined using a Fourier transform infrared spectrometer Spectrum RX 1 (Perkin Elmer), according to the carbonyl stretching absorption at 1740 cm⁻¹.[Citation20] The amount of TAG in the extract solutions was determined using a standard graph, and the amount of TAG was calculated in the dry algae (%, w/w). The experiments were performed five times, and mean values and the standard deviation were calculated.

Measurement of fatty acids composition

The fatty acid composition of the algae oil was determined using the standards EN ISO 5508 and EN ISO 5509. The analysis was performed with a Clarus 500 (Perkin Elmer) gas chromatograph. The conditions for analysis were as follows: capillary column Alltech AT-FAME (30 m–0.25 mm–0.25 µm), initial oven temperature of 210 °C held for 5 min then increased at 20 °C/min from 210 to 230 °C and held at 230 °C for 12 min. Nitrogen was used as the carrier gas. The injector temperature was 250 °C. The fatty acids were identified by comparing their retention times with the standards. The experiments were performed five times and the mean values and standard deviations were calculated.