Results and Discussion

5.2.1. Growth of N. oculata NCTU-3 aerated with different CO₂ concentration Effect of CO₂ concentration in airstream on the growth of N. oculata NCTU-3 was investigated in a batch culture incubated at 26 ± 1°C and 300 µmol m^-2 s^-1. The initial biomass inoculum was 0.01 g L^-1 (about 7 × 10⁵ cells mL^-1) and the cultures were aerated with air (CO₂ concentration is approximate 0.03%), 2, 5, 10, and 15% CO₂. The cultures were sampled at an 8-h interval. The specific growth rate was calculated from the cultures in each experiment. Figure 7 shows the microalgal growth aerated with different CO2 concentrations.

After 6-8 d, the growth of air and 2% CO2 aerated cultures were reached a plateau stage and the biomass concentration of N. oculata NCTU-3 were 0.268 ± 0.022 and 1.277 ± 0.043 g L^-1, respectively. Whereas, the growth of microalga aerated with 5, 10, and 15% CO2 were

completely inhibited. The specific growth rate in the air and 2% CO2 aerated cultures were 0.194 d^-1 and 0.571 d^-1, respectively. The culture aerated with 2% CO2 showed an optimal growth potential. When the N. oculata NCTU-3 culture aerated with 2% CO2, not only the biomass was greatly produced but also the specific growth rate was enhanced compared with those in the culture aerated with air. This result was confirmed by Hu and Gao (2003). They indicated that microalga, Nannochloropsis sp., grew best in an enriched CO2 aeration compared with air aeration. It may due to enough carbon sources for microalgal growth without carbon source limitation. The significant inhibition of high CO2 aeration, 5 to 15%, was also confirmed by the reports that the concentration of CO2 aeration above 5% could be harmful to microalgal cells and inhibit the microalgal growth (Silva and Pirt, 1984;Sung et al., 1999; Chang and Yang, 2003; de Morais and Costa, 2007b).

5.2.2. Lipid content of microalga at different growth phases

The microalgal cells from logarithmic, early stationary phase and stationary phase were collected to measure lipid content and supernatant from the collected samples was also obtained for determining the nitrate content in broth. The result showed that the lipid

accumulation in microalgal cells was associated with growth phases. The lipid content of N.

oculata NCTU-3 cells at logarithmic, early stationary phase and stationary phase was 30.8, 39.7 and 50.4%, respectively. This result indicated that lipid accumulation increases as N.

oculata NCTU-3 approaches into stationary phase. The decreased nitrate content in the broth of N. oculata NCTU-3 culture from logarithmic phase to stationary phase was found (data not shown). It is hinted that the N. oculata NCTU-3 culture from logarithmic phase to stationary phase would accompany with the nitrate depletion. Roessler (1988) reported that the nutrient deficiency induced an increase in the rate of lipid synthesis in a diatom, Cyclotella cryptica, and resulted in lipid accumulation in the cells. It is also indicated that lipid accumulation is related to nitrogen depletion as a nutrient deficiency (Roessler et al., 1994; Takagi et al., 2000). The result is confirmed by these previous reports that the microalga, N. oculata NCTU-3, shows the metabolic effect of nitrogen depletion related to the increasing lipid accumulation.

5.2.3. Effect of CO2 concentration on cell growth in semicontinuous cultures

For the study of lipid accumulation in response to higher CO2 aeration, the microalgal cells pre-adapted to CO2 were applied. In the experiment, N. oculata NCTU-3 cells were pre-adapted to 2% CO2 before the microalga was inoculated into the semicontinuous cultures.

Moreover, a high density (approximate 0.4 g L^-1) of inoculum was applied in the cultures. The semicontinuous system was operated for 8 d and the growth was stable by each day

replacement and was maintained at logarithmic growth potential. The results showed that the growth profiles of N. oculata NCTU-3 aerated with 2, 5, 10, and 15% CO2 in the

semicontinuous system were similar (Figure 8). The average specific growth rate and maximum cell density (i.e., biomass concentration) were from 0.683 to 0.733 d^-1 and from 0.745 to 0.928 g L^-1 at different concentrations of CO2 aerated cultures, respectively (Figure 8). High CO2 aeration (5-15%) may be a harmful effect on the microalgal cells growth as shown in Fig. 1. But increasing the inoculated cell density and pre-adapting to 2% CO2

culture could promote the growth capacity of microalga in the cultures aerated with higher CO₂ concentrations. The results indicated that increasing cell density and pre-adapting microalgal cells in an adequate CO₂ concentration is an alternative approach for the

application of high CO2 aeration without drastic harmful effects on microalgal cell growth.

5.2.4. Biomass and lipid productivity in semicontinuous culture

In the semicontinuous culture system, the N. oculata NCTU-3 cells were collected at the time before culture replaced each day for determination of biomass and lipid productivity.

Table 5 summarizes the biomass and lipid productivity of N. oculata NCTU-3 cultures aerated with various CO2 concentrations. As increasing CO2 concentration of aeration from 2 to 15%, both biomass and lipid productivity were generally decreasing (Table 7). It is

reported that the lipid content was increasing associated with the increasing CO2

concentration of aeration in Chlorella fusca and Phaeodactylum tricornutum cultures (Dickson et al., 1969; Yongmanitchai and Ward, 1991). The data in this study showed an inverted result may due to different microalgae species, growth condition, and medium content(Hu and Gao, 2006). Our results show that the pH of cultures with 2, 5, 10 and 15%

CO₂ aeration was maintained at pH 7.8, 7.7, 7.3 and 7.0, respectively. Yung and Mudd (1966) reported that the carbon assimilation of lipid synthesis was decreased with decrease of pH.

This may be possibly because the higher pH having higher available bicarbonate for

carboxylation of lipid synthesis. This inference supports the result that lipid accumulated in N.

oculata NCTU-3 may be mainly affected by pH and lipid content of the microalgal cultures was decreased with decrease of broth pH.

5.2.5. Optimal CO2 concentration applied in semicontinuous cultures

In the semicontinuous system, N. oculata NCTU-3 could grow well under high CO2 (up to 15% CO2) aeration, shows the potential of the microalgal culture for CO2 removal.

Therefore, the CO2 removal efficiency in the semicontinuous system cultured with N. oculata NCTU-3 was determined by the measurement of influent and effluent of CO2 airstream. The method and operation was established and described in our previous study (Chiu et al., 2008).

The amount of CO2 between influent and effluent, and CO2 removal efficiency were recorded. The CO2 concentrations in the effluent of 2, 5, 10 and 15% CO2 aerated cultures were maintained at 0.9–1.1, 3.8–4.1, 8.3–8.7 and 12.9–13.2% CO2 over 8 d cultivation, respectively. The CO2 removal efficiency in the cultures aerated with 2, 5, 10, and 15% CO2

were 47, 20, 15 and 11%, and the amount of CO₂ removal in the cultures were 0.211, 0.234, 0.350 and 0.393 g h^-1, respectively. The efficiency of CO₂ removal in the cultured aerated

with low CO2 concentration was higher than those aerated with high CO2 concentration (de Morais and Costa, 2007a; Chiu et al., 2008). The CO2 removal efficiency in a closed photobioreactor system is dependent on microalgal species, photobioreactor, and concentration of CO2 aeration (Cheng et al., 2006; de Morais and Costa, 2007a). This

assumption was confirmed by the study in Chlorella sp., the study showed more CO2 removal capacity but lower biomass productivity in a microalgal culture treated with low CO2 aeration (Chiu et al., 2008). Cheng et al. (2006) demonstrated a Chlorella vulgaris cultured

membrane-photobioreactor obtained a maximum rate of microalgal CO2 fixation at 2% of CO2 aeration. Different photobioreactors could also bring different gaseous transfer efficiency, light harvesting efficiency, and mix efficiency (Carvalho et al., 2006). In the present study, amount of CO2 removal was 0.264, 0.293, 0.438 and 0.491 g L^-1 h^-1, however, total biomass productivity was 0.480, 0.441, 0.398 and 0.372 g L^-1 d^-1 in the cultures with 2, 5, 10 and 15%

CO₂ aeration, respectively. The microalgal cultures aerated with higher CO₂ showed lower biomass productivity. This result may due to that when the microalgal cells aerated with higher CO₂, most of the CO₂ is consumed for metabolic activity and less of CO₂ is fixed to become cellular components, i.e., biomass. The higher metabolic activity may contribute to the microalgal cells to subsist on higher CO₂ stress. The results showed that the maximal CO₂ utilization efficiency was from the cultures aerated with 2% CO₂ airstreams. It is also

indicated that the optimal concentration of CO2 aeration in the system based on the efficiency of biomass and lipid productivity was 2% CO2.

5.2.6. Comparison of productive efficiencies in semicontinuous system with different culture approaches

The comparison of productive efficiencies in the semicontinuous systems in which the culture broth were replaced at an interval of 24 h (one-day replacement) or 72 h (three-day replacement) was performed. In the systems, approximate 0.4 g L^-1 of N. oculata NCTU-3 cells was inoculated and the microalgal cultures were replaced half (for one-day replacement) or three fifth (for three-day replacement) of broth with fresh medium in the semicontinuous system after the cultures aerated with 2% CO2. Figure 9 shows the stable growth profiles of N.

oculata NCTU-3 cultured with one-day and three-day replacement. In the cultures, the broth was replaced at logarithmic phase in one-day replacement and replaced before the cells reached to early stationary phase in three-day replacement. The growth profiles of both one-day and three-day replacement cultures were stable over 12 d cultivation. Table 8 shows

the biomass and lipid productivity of N. oculata NCTU-3 cells in the semicontinuous culture system with one-day and three-day replacement. The total volume of replaced broth was 4,800 mL in one-day replacement and only 1,920 mL in three-day replacement over 12 d. The lipid content of microalga in the three-day replacement was significantly higher than that in the one-day replacement culture (41% vs. 31%). However, the total biomass and total lipid yield in the three-day replacement culture were only 24% and 32% compared with those in the one-day replacement culture, respectively. It means that the culture broth being daily replacement could be more efficient not only for biomass production but also for lipid yield.

In conclusion, the total biomass and lipid yield in the semicontinuous culture operated by one-day replacement were more efficient compared with those in three-day replacement, although the N. oculata NCTU-3 cells in the three-day replacement could increase lipid accumulation because of nutrition-deficient effect.

6. Part III: The air-lift photobioreactors with flow patterning for a