4.2.1. Evaluation of cell density and biomass
Cell density and biomass were measured more easily by optical density than by direct counting of cells or by cell dry weight (Rocha et al., 2003). Therefore, relationships between optical density and cell density and optical density and cell dry weight were established by linear regression firstly (Figure 4). Optical density precisely predicted both cell density (R2 = 0.997; p < 0.001) and biomass (R2 = 0.991; p < 0.001). Therefore, the values of optical density were used to calculate the related biomass of Chlorella sp. in each experiment according the equations established.
4.2.2. Effect of CO2 on microalgal culture at different cell density
To investigate the effect of CO2 concentration on growth, Chlorella sp. in batch culture was incubated for 4 to 8 days at 26 ± 1°C and 300 µmol m-2 s-1 and aerated with different concentrations of CO2 at 0.25 vvm. Cultures were sampled when a stationary phase of growth was reached or a microalgal growth was significantly inhibited. Specific growth rate was calculated from the logarithmic growth phase over 1 to 2 days batch culture in each experiment.
As the cells grew up to plateau stage, the biomass in air, 2% and 5% CO2 aeration with low-density cells inoculum (i.e., 8 × 105 cells mL-1) were 0.537 ± 0.016 g L-1, 1.211 ± 0.031 g L-1 and 0.062 ± 0.027 g L-1, respectively. At the aeration of 2% CO2, Chlorella sp. increased most rapidly at the specific growth rate of 0.492 d-1 and the specific growth rate markedly fell to be 0.127 d-1 when the cultures were aerated with 5% CO2. The growth of Chlorella sp. at 10% and 15% CO2 aeration was almost completely inhibited; therefore the specific growth rates were not available (Figure 5A and Table 5).
In the cultures inoculated with Chlorella sp. at high-density (i.e., 8 × 106 cells mL-1), a short lag period and steep log phase was observed when the cultures aerated with 2% and 5%
CO2 compared to those of low-density inoculum. It is worth to emphasize that the biomass and specific rate at 5% CO2 aeration in high-density inoculum was 0.899 ± 0.003 g L-1 and 0.343 d-1. The values were significantly increased as compared with those in low-density inoculum. However, the growth of Chlorella sp. was inhibited after 4 days of incubation under the conditions of 10% and 15% CO2 aeration (Figure 5B and Table 5). In the 5% CO2
aerated cultures in high-density inoculum, the biomass production and specific growth rate were strongly enhanced. This enhancement may due to enrichment of available CO2 as carbon source and the culture condition under the 5% CO2 aeration would not be significantly
changed in the culture with higher cell density inoculated.
Chlorella sp. grew rapidly in a high-density culture with CO2 aeration. The result is confirmed by the report that the waste gas or CO2 tolerance of microalgae was dependent on cell density (Lee et al., 2002; Yoshihara et al., 1996; Yun et al., 1997).
4.2.3. Effect of CO2 on cell growth in semicontinuous cultivation
The semicontinuous culture was carried out in two stages. A batch culture had an initial cell density of 8 × 106 cells mL-1 (i.e., a high-density of inoculum). At 2% CO2, cell density was allowed to increase until it reached an optical density (A682) over 5 (the cell density was around 1 × 108 cells mL-1), which occurred after 6 to 8 days of incubation. After that, half of the culture broth was replaced with fresh modified f/2 medium each day and the culture was incubated with 2%, 5%, 10%, and 15% CO2 aeration. The growth of Chlorella sp. in the semicontinuous culture was constantly similar at 2%, 5%, 10%, and 15% CO2. The average specific growth rate and biomass, respectively, were 0.58 to 0.66 d-1 and 0.76 to 0.87 g L-1 after 8 days of incubation at 2 to 15% CO2 aeration. These semicontinuous cultures aerated with different CO2 concentrations were operated for 24 days. The growth of these cultures was stable on each day. These results shows that a high concentration of CO2 (10-15%) may directly introduce to a high-density Chlorella sp. culture in the semicontinuous
photobioreactor system. The high CO2 concentration did not cause harmful effects on microalgae, indicating that the CO2 can be as carbon source for the growth of a variety of photosynthetic microalgae at high-density culture. An initial high-density of the Chlorella sp.
cultures that was adapted to 2% CO2 may overcome environment stress induced by higher CO2 (10-15%) aeration.
Selection of the mutant of Chlorella sp. represents one approach to elevating CO2
tolerance of microalgae (Chang and Yang, 2003). However, growth and cell density in the cultures aerated with high levels of CO2 are still limited in the application of these mutants.
Chang and Yang (2003) have isolated Chlorella strains NTU-H15 and NTU-H25 and found that the greatest biomass produced by each strain at 5% CO2 was 0.28 g L-1 d-1. The other mutant, Chlorella strain KR-1, showed a potential biomass of 1.1 g L-1 d-1 at 10% CO2 (Sung
et al., 1999). However, increasing the cell density in the cultures or pre-adapting cells in a low concentration of CO2 are alternative approaches to increase CO2 tolerance of microalgae without effects on microalgal growth (Lee et al., 2002; Yun et al., 1997). In our
semicontinuous photobioreactors, Chlorella sp. cells that were pre-adapted to 2% CO2 not only grew into a high-density microalgal culture but also grew fast at 10% or 15% CO2. Our results confirmed these previous studies and provided a useful system that can be applied to conversion of CO2 into biomass.
4.2.4. Effect of CO2 on CO2 reduction in semicontinuous culture
Semicontinuous Chlorella sp. culture was conducted to examine the potential of CO2
reduction in the photobioreactor using a high-density culture. Prior to the photobioreactor being operated with microalgae present, the photobioreactor was emptied and operated for 1 day without microalgae to test for any abiotic removal of CO2, at 2%, 5%, 10%, and 15% CO2. During these tests, the average influent and effluent concentrations of CO2 were similar. Thus, CO2 was not removed via an abiotic mechanism.
The amount of CO2 reduced from the airstreams was estimated in the semicontinuous Chlorella sp. cultures during an 8-day period. The difference in CO2 concentration between the influent load and effluent load were monitored. All runs in each treatment and on each day were remarkably consistent and showed a similar pattern among the influent and effluent CO2
measurements. The effluent CO2 concentrations in the influent 2%, 5%, 10% and 15% CO2
treatments was maintained at 0.8~1.0%, 3.5~3.8%, 7.9~8.4% and 12.4~12.8% CO2 during 8-day operation, respectively. The average rate of CO2 reduction in cultures at 2%, 5%, 10%, and 15% CO2 in the single photobioreactor was 0.261, 0.316, 0.466 and 0.573 g h-1,
respectively. Thus, the overall efficiency of CO2 reduction in the cultures was 58%, 27%, 20% and 16%, respectively (Figure 6). Recently, de Morais and Costa (2007a) reported greater efficiency of CO2 fixation in cultures at low CO2 concentration (6%) than in cultures at high CO2 concentration (12%). The increasing retention of CO2 in a microalgal
photobioreactor also could significantly enhance the efficiency of CO2 fixation (Cheng et al., 2006). Keffer and Kleinheinz (2002) demonstrated that air dispersed in photobioreactors operated under approximately 2 seconds of air retention time removed up to 74% of CO2 from an airstreams containing 0.16% CO2. The air retention time was around 1–1.5 second in our photobioreactor; therefore, we believe that amount and efficiency of CO2 reduction can be
In the absence of microalgae, the medium pH was 7.5 in cultures aerated with air and pH dropped to about 6.4, 6.1, 5.8, and 5.6 at 2%, 5%, 10% and 15% CO2, respectively. However, pH was greater in each culture of inoculated with Chlorella sp. Average pH was 7.6, 7.4, 7.1 and 6.8 at 2%, 5%, 10% and 15% CO2, respectively. Free CO2 in culture broth containing Chlorella sp., i.e., CO2(aq), was also measured. The CO2(aq) in the cultures was stable
throughout the period of 8 days of incubation. Average CO2(aq) in cultures aerated with 2%, 5%, 10% and 15% CO2 was 575, 605, 660 and 705 ppm, respectively. These values were consistent with the changes in culture pH. The CO2(aq) concentration was generally increased with increased influent CO2 concentration; however, the result indicates the limit on the amount of CO2 that can dissolve in the culture broth. Most of the influent CO2 flowed out of the photobioreactor directly when the CO2 concentration was more than 2%.
The efficiency of CO2 removal or fixation in a closed culture system is dependent on the microalgal species, CO2 concentration, and photobioreactor (Cheng et al., 2006; de Morais and Costa, 2007a). Cheng et al. (2006) have demonstrated that CO2 removal efficiency peak (55.3%) at 0.15% CO2 and the amount of CO2 reduction (about 80 mg L-1 h-1) peaks at 1%
CO2 in a Chlorella vulgaris culture in a membrane photobioreactor. In a three serial tubular photobioreactor, 27 to 38% and 7 to 13% of CO2, respectively, was fixed by Spirulina sp. and Scenedesmus obliquus in cultures aerated with 6% CO2 aeration. In treatments of 12% CO2 aeration, CO2 fixation efficiency was only 7~17% for Spirulina sp. and 4~9% for S. obliquus (de Morais et al., 2007a). The species dependence of efficiency of CO2 removal or fixation may be due to physiological conditions of microalgae, such as potential of cell growth and ability of CO2 metabolism.
4.2.5. Effect of CO2 on lipid and biomass production in semicontinuous culture Lipid and biomass productivity in the semicontinuous Chlorella sp. cultures were determined before the culture broth was changed each day. Table 6 summarizes the results, lipid and biomass productivity, collected from the single photobioreactor cultures under different CO2 aeration. As a daily 50% culture broth replaced in the 800 mL semicontinuous photobioreactor aerated with 2%, 5%, 10%, and 15% CO2, the total biomass and lipid productivity per day (400 mL of waste broth was recovered for measurement) of each photobioreactor was 0.422 g d-1, 0.393 g d-1, 0.366 g d-1 and 0.295 g d-1, and 0.143 g d-1, 0.130 g d-1, 0.124 g d-1 and 0.097 g d-1, respectively. In the single semicontinuous culture, both of lipid and biomass productivity decreased when the aerated CO2 concentration was
increased. However, lipid content in the cells cultured at 2%, 5%, 10%, and 15% CO2 were very similar (approximately 32~34% of dry weight). In the semicontinuous culture, the optimum condition for biomass productivity was at 2% CO2 aeration and lipid content was not affected even at high CO2 aeration. Biomass productivity at 15% CO2 aeration was 68%
of that at 2% CO2 aeration. However, our results still show the potential growth of microalgal Chlorella sp. for lipid and biomass productivity in the semicontinuous system even the cells were cultivated in the condition aerated with 15% CO2.
The lipid content of Chlorella fusca and Phaeodactylum tricornutum increased when cells were grown at increasingly higher concentrations of CO2 (Dickson et al., 1969;
Yongmanitchai and Ward, 1991). Our result was not consistent with these previous studies.
Such divergent results for lipid content of microalgae cultured under CO2 aeration may be due to differences in microalgal species, content of culture medium, and culture condition.
4.2.6. Performances of six-parallel photobioreactor system
The efficiency of CO2 removal from airstreams by Chlorella sp. was compared between the single photobioreactor and the six-parallel photobioreactor. The effects of varying CO2 concentration on growth of Chlorella sp. was similar between the single and the six-parallel photobioreactors (data not shown). In a total volume of 4,800 mL (i.e., 6 × 800 mL) of the six-parallel photobioreactor, the total amount of CO2 reduced was 1.563, 2.058, 2.757 and 3.441 g h-1 at 2%, 5%, 10% and 15% CO2 aeration, respectively (Figure 6). Thus, the amount of CO2 that reduced in the six-parallel photobioreactor was approximately six times greater than the amounts in the single photobioreactor. Therefore, the efficiency CO2 reduction in the six-parallel photobioreactor and in the single photobioreactor was also similar (Figure 6).
Daily recovery of lipid and biomass in the six-parallel photobioreactor were determined.
In each case, the amount of lipid and biomass recovered daily in the six-parallel photobioreactor was around six times greater than the amounts recovered in the single photobioreactor (Table 6). CO2 reduction efficiency and cell growth in both photobioreactor systems also were similar.
When microalgal cells grew in a closed photobioreactor, light decreases exponentially with the distance from light source (Suh and Lee, 2003). It will be a problem for diameter of scale-up photobioreactor with external lighting. Our results show that our photobioreactor could be extended to parallel multiple units of photobioreactor for discharging waste gas in a
large scale without decreasing biomass and lipid productivity, and efficiency of CO2 reduction.
Additionally, increasing the length of tubular photobioreactor and gas sparging into small bubbles can be considered in a scale-up system. Longer tubular photobioreactor and small bubbles could increase the retention time of gas in photobioreactor and the bubbles absorbed into cultures, and then increases the efficiency of CO2 reduction.
5. Part II: Biomass production and CO2 utilization of Nannochloropsis