2.1 Microalgae strain
The microalgae Chlorella sp. TT-1 was originally obtained from the collection of Taiwan Fisheries Research Institute (Tung-Kang, Ping-Tung, Taiwan) and isolated in our laboratory by chemical mutagenesis.
The species of Chlorella sp. isolated in Taiwan was unidentified. However, the partial sequence of 18S rRNA (599 bp) of the Chlorella sp. has been amplified and sequenced for species identification in this study. This result of sequence alignment was performed by NCBI nucleotide blast [Wu et al., 2001].
2.2 Culture medium and chemicals
Chlorella sp. TT-1 was cultured in artificial sea water enriched with f/2 medium and an illumination of 300 mol/m2/s by white fluorescent light at 26 ± 1℃. Artificial sea water has following composition (per liter): including 29.23 g NaCl (Showa, Tokyo, Japan), 1.105 g KCl (Showa), 11.0862 g MgSO4 . 7H2O (Amresco, Solon, OH, USA), 1.21 g Tris-base (Merck, Darmstadt, Germany), 1.83 g CaCl2 . 2H2O (Amresco), 0.25 g NaHCO3 (Amresco). f/2
medium has following composition (per liter): 75 mg NaNO3 (Showa), 5 mg NaH2PO4 . H2O (Sigma, Saint Louis, MO, USA), 1 mL of trace metal solution, and 1 mL of vitamin solution [Guillard, 1975]. Trace elemental solution (per liter) includes 4.36 g Na2 . EDTA (Amresco), 3.16 g FeCl3 . 6H2O (Sigma), 180 mg MnCl2 . 4H2O (Sigma), 10 mg CoCl2 . 6H2O (Sigma), 10 mg CuSO4 .
5H2O (Sigma), 23 mg ZnSO4 .
7H2O (Showa), 6 mg Na2MoO4 (Sigma). Vitamin solution (per liter) includes 100 mg vitamin B1 (Sigma), 0.5 mg vitamin B12 (Sigma) and 0.5 mg biotin (Sigma).
The microalgae were selected for the studies of CO2 challenge and the high biomass concentration which were cultured in modified f/2 medium in artificial sea water at 26 ± 1 ℃ with an illumination of 300 mol/m2/s by white fluorescent light. Modified f/2 medium has following composition (per liter): including 225 mg NaNO3 (Showa), 15 mg NaH2PO4 . H2O (Sigma, Saint Louis, MO, USA), 3 mL of trace metal solution, and 1 mL of vitamin solution
2.3 Experimental system of indoor photobioreactor
The microalgae cells were cultured in photobioreactors with a working volume of 1 L [Chiu et al., 2008]. The photobioreactors were placed in an incubator at 28 ± 1℃ with a surface light intensity of approximately 300 mol/m2/sprovided by continuous, cool-white, fluorescent lights. The photobioreactor was a cylindrical glass column which the diameter and length of the photobioreactor was 5 cm and 80 cm respectively. The gas was supplied from the bottom of the photobioreactor. The flue gas was collected from coke oven in China Steel Corporation, and was exhausted into tank which offered the space to mix it with air. Different flue gas and air volume mixed in the tank would form different ratio gases which would be introduced into bioreactors and utilized by microalgae as nutrient. The gas flow rate was adjusted by using a gas flow meter (Dwyer Instruments, Michigan, IN, USA). The evaluation of tolerance to the flue gas in microalgae cultures, and the microalgae cells in each treatment were sampled every 24 hr for determination of the biomass concentration. The figure about experimental system is presented in Figure 2-1.
2.4 Preparation of the inoculums
A stock culture of Chlorella sp. (approximately 1 × 105 cells/mL) was incubated in an Erlenmeyer flask containing 800 mL working volume of modified f/2 medium at 26 ± 1℃ and 300 mol/m2/s. After Six days culture, the microalgae cells were harvested by
centrifugation at 3,000 × g for 5 min, after which the pelleted cells were resuspended in 50 mL fresh modified f/2 medium. The density of cells in the culture was then measured and the cells were separated for the further experiments.
2.5 Experiment design
RSM based on central composite design (CCD) was applied to optimize the experimental conditions for the microalgae cultivation, lipid content, and the compositions of FAME
content. Three critical independent parameters affecting microalgae cultivation, lipid content, and the compositions of FAME content: flue gas ratio(x1), aeration rate(x2), and initial density(x3) were selected as the independent variables based on the experiments.
Experimental range and levels of independent variables for microalgae biomass
productivity, lipid content, and the compositions of FAME content were presented in Table 3-7 and Table 3-8.
In the optimization process, the responses can be simply related to chosen variables by linear or quadratic models. A quadratic model, which also includes the linear model, is given below:
β β β β β β β β β
β ε
Where Y is the response and x1, x2, and x3 are the independent variables effects. x1, x2, and x3 are the square effects. x1x2, x1x3, and x2x3 are the interaction effects. β1, β2, andβ3 are the linear coefficients. β4, β5, andβ6 are the interaction coefficients. β7, β8, andβ9 are the squared coefficients. β0 and ε are the constant and the random error respectively. This model is preferred because a relatively few experimental combinations of the independent variables are adequate to estimate potentially complex response function.
The calculations were based on a least squares analysis. Using this technique, two assumptions are made [Spolaore, 2006b]:
The expected value of the random error is zero, and its variance is constant over the range of the experimental factors that are used to collect data.
There is no association of the random error for any one data point with the random error for any other data points.
In the experiment design total twenty experiments were performed in randomized order as required in many design procedures. The design consist of fractional factorial design including the three variables at two levels each augmented by six star points, which α is the distance of the star point from the center, and six center points to evaluate the pure error.
Experiment layout and result were analyzed using Design Expert 8.0.6 software and a
regression model was proposed. Analysis of Variance (ANOVA) was based on the proposed.
2.6 Lipid extraction
Lipid extraction was according to the modified method previously reported [Chiu et al., 2009b]. The microalgae cells were centrifuged and washed with deionized water twice, and obtained the dry biomass by lyophilization. The dried sample (200 mg) was mixed with methanol/chloroform solution (2/1, v/v) and sonicated for 1 hr. The mixture with
methanol/chloroform solution was precipitated and added chloroform and 1 % NaCl solution to give a ratio of methanol, chloroform, and water of 2:2:1. The mixture was centrifuged and the chloroform phase was recovered. Finally, the lipids were weighted after chloroform was removed under vacuum by a rotary evaporator.
2.7 Transesterification
The extracted oil samples were placed in a glass test tube and mixed with 4.0 mL chloroform, 3.4 mL methanol, and 0.6 mL sulfuric acid. The samples were sonicated for 60 minutes. After the reaction was completed, the tubes were removed from the water bath and allowed to cool to room temperature. Then, 2 mL distilled water was added to the tube and thoroughly mixed using a vortex. The samples were allowed to separate, forming a biphasic solution. The organic layer containing FAME was collected and transferred to a pre-weighed glass vial. The solvent was then evaporated using N2 and heated at 70 ℃ for 40 min. Finally the mass of FAME was determined via weighing.
2.8 Fatty acid profile analysis
The fatty acid composition was determined FOCUS Gas Chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an flame ionization detector (FID) and trace GC capillary column (Thermo Fisher Scientific, Waltham, MA, USA), which was a
cyanopropylphenyl based phase specifically designed for the separation of FAMEs. A 30 m long column was used with a diameter of 0.32 mm and a 0.25 μm thick film. The amount of sample injected was 2 μL. The stripping gas was nitrogen at a flow rate of 1.3 mL/min, and the injector and detector temperatures were 250 and 280 ℃ respectively. The initial column temperature was 150 ℃ where it remained for 1 min, then rising from 150 to 180 ℃ at
10 ℃/min, remaining at 180 ℃ for 3 min, then rising from 180 to 220 ℃ at 1.5 ℃/min, remaining at 220 ℃for 1 min, and finally rising from 220 to 260 ℃ at 30 ℃/min, remaining at 260 ℃ for 5 min. The fatty acids were identified by comparison of the retention times with those of the standards using the software Chrom-Card Data System (Thermo Fisher Scientific, Waltham, MA, USA). The analysis of GC profile about the FAME transesterificated from the lipid in microalgae is shown in Figure 2-2.
2.9 Analyses
2.9.1 Microalgae cell counting
A direct microscopic count was performed on the sample of microalgae suspension using a Brightline Hemacytometer (BOECO, Hamburg, Germany) and a Nikon Eclipse TS100 inverted metallurgical microscope (Nikon Corporation, Tokyo, Japan).
2.9.2 Measurement of growth rate
Biomass concentration (dry weight per liter) of cultures were measured according to the method reported previously [Chiu et al., 2009a]. Regression equations of the relationship between optical density and cell dry weight were established and shown as follows:
y = 0.2529 x – 0.0153 R² = 0.9898
The value y is biomass concentration (g/L). This value was determined according the method previously reported [Chiu et al., 2009a]. Microalgae cells were collected, centrifuged and washed with deionized water. The washed microalgae pellet was dried at 105 ℃ for 16 hr;
afterward, the dried cells were for dry weight measurement. The value x1 is optical density measured by the absorbance at 682 nm (A682) in an Ultrospec 3300 pro UV/Visible
spectrophotometer (Amersham Biosciences, Cambridge, UK). Each sample was diluted to give an absorbance in the range of 0.1–1.0 if optical density was greater than 1.0.
The optical density was used to evaluate the biomass concentration of Chlorella sp. TT-1 in each experiment. In the present study, we used biomass concentration (g/L) for the
quantification of Chlorella sp. TT-1 cell density in the culture. The biomass productivity was measured and according the equation showed as follows:
Where Wf and Wi is the final and initial biomass concentration, respectively. ∆t is the cultivation time in days.
2.9.3 Measurements of pH
Sample pH was determined directly with an ISFET pH meter KS723 (Shindengen Electric Mfg.Co.Ltd, Tokyo, Japan). The pH meter was calibrated daily using pH 4 and 7 solutions.
2.9.4 Measurement of light
Light intensity was measured from the light-attached surface of the photobioreactor using a Basic Quantum Meter (Spectrum Technologies, Plainfield, IL, USA).
2.9.5 Determinations of CO2(g)
The CO2 concentration in airstreams, CO2(g), was measured using a Guardian Plus Infra-Red CO2 Monitor D-500 (Edinburgh Instruments Ltd, Livingston, UK).