3.1. Microalgal cultures, medium and chemicals
A culture of Chlorella sp. was obtained from Taiwan Fisheries Research Institute (Tung-Kang, Taiwan). 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. The result of sequence alignment was performed by NCBI nucleotide blast (Wu et al., 2001). The result indicates that the Chlorella sp. is identified as several Chlorella sp. strain, such as KAS001, KAS005, KAS007, KAS012, MBIC10088, MDL5-18 and SAG 211-18. The microalga N. oculata NCTU-3 was originally obtained from the collection of Taiwan Fisheries Research Institute (Tung-Kang, Taiwan) and screened for its potential ability of growth and biomass production at National Chiao Tung University, Taiwan (data not shown). The cells of Chlorella sp. and N. oculata were cultured in the modified f/2 medium in artificial sea water (per liter), including 29.23 g NaCl, 1.105 g KCl, 11.09 g MgSO4 ·7H2O, 1.21 g Tris-base, 1.83 g CaCl2 ·2H2O, 0.25 g NaHCO3, and 3.0 mL of trace metal solution (Guillard, 1975). The trace metal solution (per liter) contains 281.3 mg NaNO3, 21.2 mg NaH2PO4 ·H2O, 16.35 mg Na2 ·EDTA, 11.8 mg FeCl3 ·6H2O, 675 μg MnCl2 ·4H2O, 37.5 μg CoCl2 ·6H2O, 37.5 μg CuSO4 ·5H2O, 82.5 μg ZnSO4 ·7H2O, 22.5 μg Na2MoO4, 0.375 mg vitamin B1, 0.188 μg vitamin B12 and 0.188 μg biotin.
3.2. Experimental system with photobioreactor
The microalga was incubated in a cylindrical glass reactor (30 cm length, 7 cm diameter) with 800 mL of working volume. The photobioreactor for microalgal culture and CO2
reduction is presented schematically in Figure 2. Cultures were placed on a bench at 26 ± 1°C under continuous, cool white, fluorescent light. Light intensity was approximately 300 mol m-2 s-1 at the surface of the photobioreactor. Filtered (0.22 m) ambient air was mixed with CO2 to give concentrations of CO2 of 2%, 5%, 10%, and 15%. Cultures in the photobioreactor were aerated continuously with one of the mixtures at a rate of 200 mL min-1 (i.e., 0.25 vvm, volume gas per volume media per min).
3.3. Preparation of the inoculum
A stock culture of microalgal cells (approximately 1 × 105 cells mL-1) was incubated in an Erlenmeyer flask containing 800 mL working volume of modified f/2 medium at 26 ± 1°C and 300 mol m-2 s-1. Six days after inoculation, microalgal cells were harvested by
centrifugation at 3,000 × g for 5 min, after which the pelleted cells were resuspended in 50 mL modified f/2 medium. The density of cells in the culture was then measured and the cells were separated for the further experiments.
3.4. Experimental design of indoor batch cultivation
The photobioreactor was filled with 750 mL modified f/2 medium. The medium was aerated for 24 hours and then inoculated with 50 mL of precultured Chlorella sp. containing either 8 × 105 cells mL-1 (low-density) or 8 × 106 cells mL-1 (high-density). The cells from a 50 mL (at the density of 3.2 × 107 cells mL-1) of precultured micralgal Chlorella sp. were subcultured into the 800 mL culture photobioreactor as low-density and the tenfold concentrated micralgae by centrifugation were subcultured into the photobioreactor as
high-density culture. Air of different CO2 concentration was produced by mixing air and pure CO2 at 0.25 vvm. Each air/CO2 mixture was adjusted to desired concentration of 2%, 5%, 10%, and 15% CO2 in airstreams. Cultures were incubated for 4-8 days. Every 8 hours, each culture was sampled to determine optical density, microalgal dry weight, and culture pH.
3.5. Experimental design of indoor semicontinuous cultivation
The semicontinuous cultivation system was setup in a single photobioreactor and a system with six-parallel photobioreactor. Each unit of photobioreactor contained 800 mL cultured microalgae. The culture was started as a batch culture. Precultured microalgae were inoculated into the photobioreactor under 2% CO2 aeration. When cell density reached about 1 × 108 cells mL-1 (the value of A682 > 5), half of volume of the culture broth was replaced with fresh modified f/2 medium every 24 hours. In each photobioreactor, the culture was aerated with 2%, 5%, 10% and 15% CO2 at 0.25 vvm. Before fresh medium was added, the culture broth was sampled to estimate optical density, microalgal dry weight, lipid content, and pH. The amount of CO2 reduced from the airstreams was estimated from the difference between the CO2 concentrations in influent and effluent airstreams of the photobioreactors.
For the maintenance of biomass production, a semicontinuous culture mode was applied.
The microalgal cells were pre-cultured in a batch and fed-batch culture until reaching
approximately 5 g L-1. After the pre-culture, the cultures were replaced with ratios of one half (1/2), one third (1/3) and one fourth (1/4) of fresh medium. Each replacement was executed when the microalgal cultures had grown back to approximately 5 g L-1. The semicontinuous cultures were operated for at least two cycles of replacement (at least 15 days). The biomass productivity for each replacement was evaluated when the biomass concentration had again reached approximately 5 g L-1. The obtained biomass from each replacement was also determined from the replaced broth.
3.6. Microalgal cell counting and dry weight
A direct microscopic count was performed on the sample of microalgal suspension using a Brightline Hemacytometer (BOECO, Hamburg, Germany) and a Nikon Eclipse TS100 inverted metallurgical microscope (Nikon Corporation, Tokyo, Japan). Cell density (cells mL-1) was 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 due to the biomass will be underestimated when the optical density is out of the linear range. Therefore, the sample was diluted to measure getting an absorbance in the range 0.1–1.0. Microalgal dry weight (g L-1) was measured according to the method previously reported (American Public Health Association, 1998).
Culture broth of samples was removed by centrifugation and washed twice with deionized water. Finally, the microalgal pellet was collected from the deionized water by centrifugation.
Dry weight was measured after drying the microalgal pellet at 105°C for 16 hours (Takagi et al., 2006).
3.7. Measurement of growth rate
The optical density of microalgal cells was converted into dry weight per liter of culture by a regression equation. Biomass was calculated from microalgal dry weight produced per liter (g L-1). Specific growth rate (μ, d-1) was calculated from
where Wf and W0 were the final and initial biomass concentration, respectively. △t was the
cultivation time in day (Ono and Cuello, 2007). Thus, we used biomass (g L-1) to quantify microalgal cells in culture.
3.8. pH and light measurements
Sample pH was directly determined using an ISFET pH meter KS723 (Shindengen Electric Mfg.Co.Ltd, Tokyo, Japan). The pH meter was calibrated daily using pH 4 and 7 solutions. Light intensity was measured adjacent to the bioreactor at liquid level using a Basic Quantum Meter (Spectrum Technologies, Inc., Plainfield, IL).
3.9. Lipid extraction and measurement
For the lipid extraction, the microalgal cells were obtained by centrifuging a 50-mL sample of culture at 3,000 × g for 15 min. The cells were washed with deionized water twice, lyophilized, and weighed. A sample (30 mg) was precipitated in methanol/chloroform (2/1, v/v) and sonicated for 1 hour. Chloroform and 1% NaCl were added to give mixture to a ratio of methanol, chloroform, and water of 2:2:1. The mixture was centrifuged at 1,000 × g for 10 min and the chloroform phase was collected. Chloroform was evaporated under vacuum in a rotary evaporator to remove organic solvent. The remaining from the evaporation was weighed as lipid (Takagi et al., 2006).
3.10 Measurement of lipid content by fluorescent spectrometry
For fast determination of lipid content, a fluorescent spectrometric method was applied.
In the method, the microalgal cells were stained with Nile Red (Sigma, St. Louis, MO, USA) followed the protocol reported by de la Jara et al. (2003). In brief, 1 mL of 1 × 106 cells suspension was added 50 μL of Nile Red in acetone working solution as a concentration of 0.1 mg mL−1 for lipid staining. The mixture was gently inverted for mixing and incubated at 37°C in darkness for 10 min. In the detection, the fluorometer with a 480 nm excitation filter and a 580 nm emission filter was used. Non-stained cells were used as an auto-florescence control. The relative florescence intensity of Nile Red was calculated as florescence intensity of Nile Red stained subtracted auto-florescence intensity signal (Lee et al., 1998; Liu et al., 2008). The following equation of the correlation curve indicated fluorescent intensity of Nile Red staining vs. lipid content measured by gravimetric method.
y = 1.680x + 5.827 R2 = 0.994 (p < 0.001)
The value y is total lipid content determined by gravimetric method. The value x is the relative arbitrary unit obtained Nile Red fluorescent spectrometric method.
3.11 Measurement of medium nitrate content
Depletion of nutrients in microalgal culture was monitored by the determination of medium nitrate content (Tonon et al., 2002). It is a simple method for investigating the adequate content of nutrients for microalgal culture in the semicontinuous cultivation. Nitrate concentration was determined according to the method reported by Collos et al. (1999). A sample collected from photobioreactor was centrifuged at 3,000 × g for 5 min. The
supernatant was collected and the absorbance was measured at 220 nm. Authentic sodium nitrate at a concentration of 0 to 440 μM was used as a standard.
3.12 Determinations of CO2(g) and CO2(aq)
The CO2 concentration in airstreams, CO2(g), was measured using a Guardian Plus Infra-Red CO2 Monitor D-500 (Edinburgh Instruments Ltd, Livingston, UK). Free CO2 in the aqueous solution, CO2(aq), was measured by a HANNA Carbon Dioxide Test Kit (KI 3818;
Hanna Instruments, Woonsocket, RL).
3.13 Photobioreactors and operation of microalgal culture
Three types of photobioreactor were designed and used in this study: (i) without inner column (i.e. a bubble column), (ii) with a centric-tube column and (iii) with a porous centric-tube column (Figure 3). The working volume in the photobioreactors was 4 L. The gas supply is from the bottom of the photobioreactor. The photobioreactors were placed in an incubator at 26±1°C, with a light intensity of approximately 300 mol m-2 s-1 at the surface of the photobioreactor provided by a continuous, cool white, fluorescent light source. The outer column with a diameter of 100 mm was made of glass, and the inner column with a diameter of 45 mm was made of acrylics. The heights of the outer glass column and the inner acrylic column were 650 and 600 mm, respectively. The dimensions of the three photobioreactors are also shown in Figure 3. The flow pattern was determined through the dye technique. Batch
cultures were used to inoculate the three types of photobioreactor (without inner column, with centric-tube column, and with porous centric-tube column) at an initial biomass concentration of 1.0 g L-1, and the cultures were aerated with 5% CO2 at an aeration rate of 1.0 L/min (i.e.
0.25 vvm).
3.14 Determination of CO2 removal efficiency
The CO2 concentration in the airstreams was sampled as influent and effluent load and measured using a Guardian Plus Infra-Red CO2 Monitor D-500 (Edinburgh Instruments, Livingston, UK). The efficiency of CO2 removal was determined by the difference of the CO2 concentration between the influent and the effluent load of the photobioreactor with the microalgal culture. The removal efficiency (%) was determined by the following formula:
2 2
2
Influent of CO Effluent of CO Influent of CO 100%
A comparison of the CO2 removal efficiencies of microalgal cultures with different aeration rates and microalgal cell densities (i.e. different biomass concentrations) was performed. The specific concentration of CO2 gas was provided by a commercial premixed-gas steel cylinder.
The gas flow rate was adjusted with a gas flow meter (Dwyer Instruments, Michigan City, IN, USA) to give a flow rate of 0.125 vvm, 0.25 vvm, and 0.5 vvm.
3.15. Experimental system of indoor photobioreactor for on-site bioremediation experiments
The microalgal cells were cultured in photobioreactors with a working volume of 800 mL (Chiu et al., 2008). The photobioreactors were placed in an incubator at 25 ± 1°C with a surface light intensity of approximately 300 mol m–2 s–1 provided by continuous, cool-white, fluorescent lights. The photobioreactor was made of glass, and the diameter of the
photobioreactor was 70 mm. The gas was supplied from the bottom of the photobioreactor.
The CO2-enriched gas was premixed with air and pure CO2 for the flue gas experiments as a control gas. In the gas airstream, CO2 concentration was adjusted to 2, 10 and 25% for cultures as control experiments. The flue gas (approximately 25% CO2, 4% O2, 80 ppm NO and 90 ppm SO2) was collected from coke oven in China Steel Corporation and was directly introduced into microalgal cultures. The gas flow rate was adjusted to 0.05 vvm (volume gas
per volume broth per min) using a gas flow meter (Dwyer Instruments, Inc., Michigan city, IN, USA). The evaluation of tolerance to the flue gas in microalgal cultures, initial biomass concentration of Chlorella sp. MTF-7 cultures were approximately 0.2 g L–1. The microalgal cells in each treatment were sampled every 24 h for determination of the biomass
concentration.
3.16.Experimental system of outdoor photobioreactor
The outdoor photobioreactor was cylindrical and made of acrylic polymer. The column was 300 cm in length and 16 cm in diameter. The working volume of the photobioreactor was 50 L (Ong et al., 2010). The gas flow rate was adjustable using a gas flow meter. The source of flue gas was from a coke oven in China Steel Corporation (Kaohsiung, Taiwan). The concentrations of CO2, , O2, NO and SO2 in the flue gas were 23 ± 5%, 4.2 ± 0.5%, 78 ± 4 ppm and 87 ± 9 ppm (Oct. 1 – Nov. 15, 2010), respectively. In the intermittent flue gas aeration, culture aeration was controlled by a gas switch, and a gas-switching cycle was performed with a flue gas inlet load for 30 min followed by air inlet load for 30 min (30 min flue gas/30 min air) during the day. The inlet and outlet loads were real-time monitored by a gas analyzer (AMETEK, Inc., Paoli, PA, USA) to determine the concentrations of CO2, O2, NO and SO2.
3.17. Chemical analyses
In the on-site bioremediation experiments, the inlet and outlet loads of airstreams were real-time monitored by a gas analyzer. The concentration of O2, CO2, NO and SO2 in flue gas were measured using Landcom III portable gas analyzer (AMETEK, Inc., Paoli, PA, USA).
3.18 Statistics
All values are expressed as mean standard deviation (SD). A Student’s t test was used to evaluate differences between groups of discrete variables. A value of P < 0.05 was considered statistically significant.
4. Part I: Reduction of CO2 by a high-density culture of Chlorella sp. in a