Results and Discussion - 建立一光生物反應系統用於微藻的高密度養殖與二氧化碳的減量

3.1. Evaluation of cell density

Nannochloropsis oculata (R² = 0.995; p < 0.001) (Figure 3-2), Skeletonema costatum (R² = 0.998; p < 0.001) (Figure 3-3), Isochrysis aff. Galbana (R² = 0.992; p < 0.001) (Figure 3-4) and Tetraselmis chui (R² = 0.993; p < 0.001) (Figure 3-5), respectively. Therefore, the values of optical density were used to calculate the related cell density of Chlorella sp., Nannochloropsis oculata, Skeletonema costatum, Isochrysis aff. galbana and Tetraselmis chui in each experiment according the equations established in this study.

3.2. The microalgae was screened for its potential ability of growth and biomass production

We attempted to study the growth capacities of microalgae via the controlling cultural environment and medial nutrition. Five microalgae, Chlorella sp., Nannochloropsis oculata, Skeletonema costatum, Isochrysis galbana and Tetraselmis chui, were used in this study.

Figure 3-6 was screened growth potential and biomass concentration of the microalgae. The optimal growth potential of Chlorella sp., Nannochloropsis oculata, Skeletonema costatum, Isochrysis galbana and Tetraselmis chui cultured in the f/2 medium at 26 ± 1°C and 300 μmol/m²/s for 24hr lighting with 0.25 vvm air aeration (designed as normal cultural medium) with air aeration were 1.55, 1.51, 0.5, 0.72 and 0.99 d^-1, respectively. The maximum cell density of Chlorella sp., Nannochloropsis oculata, Skeletonema costatum, Isochrysis galbana and Tetraselmis chui cultured in the f/2 medium (designed as normal cultural medium) with air aeration were 4.7 × 10⁷, 5.5 × 10⁷, 1.9 × 10⁷, 2.1 × 10⁷,and 1.9 × 10⁷ cells/mL, respectively.

Chlorella sp. and Nannochloropsis oculata were selected from the study because of the higher growth potential and cell density. And then Chlorella sp. and Nannochloropsis oculata were

selected for the studies of CO2 challenge and the high biomass concentration.

3.3. Evaluation of biomass concentration

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 dry weight were established by linear regression firstly. Optical density precisely predicted biomass Chlorella sp. (R² = 0.992; p < 0.001) (Figure 3-7) and Nannochloropsis oculata (R² = 0.999; p < 0.001) (Figure 3-8). Therefore, the values of optical density were used to calculate the related biomass of Chlorella sp., Nannochloropsis oculata, in each experiment according the equations established in this study.

3.4. Chlorella sp. culture at different cell density aerated with different CO

concentration

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/m²/s for 24hr lighting 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 concentration in air, 2% and 5% CO2

aeration with low-density biomass inoculum 0.01 g/L (i.e., 8 × 10⁵ cells/mL) were 0.537 ± 0.016, 1.211 ± 0.031, and0.062 ± 0.027 g/L, respectively. Areation of air (CO2

concentration is approximate 0.03%), 2, 5, 10 and 15% CO2, the microalgae culture medium pH was 9.8, 7.8, 6.5, 6.1, 5.8, respectively. At the aeration of 2% CO2, Chlorella sp.

increased most rapidly at the specific growth rate of 0.492 μ, and the specific growth rate markedly fell to be 0.127 μ when the cultures were aerated with 5% CO2. The growth of Chlorella sp. at 10% and 15% CO2 aeration was almost completely inhibited and low pH;

therefore; the specific growth rates were not available (Figure 3-9A and Table 3-1). At 2%

CO2, growth of Chlorella sp. became stable after 6-8 days of incubation. Optical density at A682 was greater than 5 and biomass was greater than 1.0 g/L.

In the cultures inoculated with Chlorella sp. at high-density 0.1 g/L (i.e., 8 × 10⁶

cells/mL), 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 pH, maximum biomass concentration and specific rate at 5% CO2 aeration in high-density inoculum was 7.6, 0.899 ± 0.003 g/L and 0.343 μ, respectively. The values were

significantly increased as compared with those in low-density inoculum. However, the growth of Chlorella sp. was inhibited after 3 days of incubation under the conditions of 10%

and 15% CO2 aeration (Figure 3-9B and Table 3-1). At 2% and 5% CO2, a short lag period was observed in cultures with high-density inoculum but not in cultures with low-density inoculum. Chlorella sp. grew slowly at 10% and 15% CO2. Optical density at A682 was less than 1.5. Moreover, at 10% and 15% CO2, growth was inhibited after 3 days of incubation.

After 6 days of incubation at 2% CO2, growth reached a plateau and biomass was over 1.3 g/L.

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 reports that the waste gas or CO2 tolerance of microalgae was dependent on cell density [Yoshihara et al., 1996; Yun et al., 1997; Lee et al., 2002].

3.5. Nannochloropsis oculata culture at different cell density aerated with different CO

concentration

Effect of CO2 concentration in airstream on the growth of Nannochloropsis oculata was investigated in a batch culture incubated at 26 ± 1°C and 300 µmol/m²/s for 24 hr lighting and with 0.25 vvm aeration. The initial biomass inoculum was 0.01 g/L (about 7 × 10⁵ cells/mL) 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 3-10 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

Nannochloropsis oculata were 0.268 ± 0.022 and 1.277 ± 0.043 g/L, respectively. The final pH was 9.8, 7.7, 6.4, 6.1 and 5.8 at air, 2, 5, 10, 15% CO2 aeration, 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 μ and 0.571 μ,

respectively. The culture aerated with 2% CO2 showed an optimal growth potential. When the Nannochloropsis oculata 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].

3.6. Effect of CO

on Chlorella sp. in semi-continuous cultivation

3.6.1. Effect of CO₂ on cell growth in semi-continuous culture

The semi-continuous culture was carried out in two stages. A batch culture had an initial cell density of 8 × 10⁶ cells/mL (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), 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 semi-continuous culture was constantly similar at 2, 5, 10, and 15% CO2 (Figure 3-11).

The average specific growth rate and biomass, respectively, were 0.58 to 0.66 μ and 0.76 to 0.87 g/L after 8 days of incubation at 2% to 15% CO2 aeration. These semi-continuous 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

semi-continuous 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/d. The other mutant, Chlorella strain KR-1, showed a potential biomass of 1.1 g/L/d 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 [Yun et al., 1997; Lee et al., 2002]. In our

semi-continuous 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.

3.6.2. Effect of CO₂ on biomass production in semi-continuous culture

Biomass productivity in the semi-continuous Chlorella sp. cultures were determined before the culture broth was changed each day. Table 3-2 summarizes the results, biomass productivity, collected from the single photobioreactor and the six-parallel photobioreactor cultures under different CO2 aeration. As a daily 50% culture broth replaced in the 800 mL semi-continuous photobioreactor aerated with 2, 5, 10, and 15% CO2, the total biomass productivity per day (400 mL of culture broth was recovered for measurement) of each photobioreactor was 0.421, 0.404, 0.366 and 0.361 g/L/d, respectively. In the single

semi-continuous culture, biomass productivity decreased when the aerated CO2 concentration was increased. In the semi-continuous culture, the optimum condition for biomass

productivity was at 2% CO2 aeration and 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 biomass productivity in the semi-continuous system even the cells were cultivated in the condition aerated with 15% CO2.

3.7. CO

reduction by Chlorella sp. in semi-continuous culture

Semi-continuous 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 difference in CO2 concentration between the influent load and effluent load were monitored in the semi-continuous Chlorella sp. cultures during an 8-day period on 300 µmol/m²/s for 24hr lighting at 26 ± 1°C (Figure 3-12). The influent and effluent CO2

concentrations in each culture were measured at 6, 12, and 18 hr after the cultured broth was replaced each day. 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/hr, respectively (Figure 3-13). Thus, the overall efficiency of CO2 reduction in the cultures was 58, 27, 20 and 16%, respectively (Figure 3-14). Recently, de Morais and Costa [2007b] reported greater efficiency of CO2

reduction 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 reduction [Cheng et al., 2006]. Keffer and Kleinheinz [2002] demonstrated that air dispersed in photobioreactors operated under

approximately 2 sec 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 sec in our photobioreactor;

therefore, we believe that amount and efficiency of CO2 reduction can be improved by

increasing the CO2 retention time in the photobioreactor system.

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.. Initial culture medium pH was between 8.0-8.2. Stably average pH was 7.6, 7.4, 7.1 and 6.8 at 2, 5, 10 and 15% CO2

aeration, respectively. Free CO2 concentration 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 reduction in a closed culture system is dependent on the microalgal species, CO2 concentration, and photobioreactor [Chen et al., 2006; de Morais and Costa, 2007]. Cheng et al. [2006] have demonstrated that maximum CO2 removal efficiency (55.3%) at 0.15% CO2 and the maximum CO2 reduction rate (about 80 mg/L/hr) 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 reduction efficiency was only 7–17% for Spirulina sp. and 4–9% for S. obliquus [de Morais and Costa, 2007]. The species dependence of efficiency of CO2

removal or reduction may be due to physiological conditions of microalgae, such as potential of cell growth and ability of CO2 metabolism.

3.8. Performance of six-parallel photobioreactor system with Chlorella sp.

The efficiency of CO2 removal from airstreams by Chlorella sp. was compared between the single photobioreactor and the six-parallel photobioreactor. Both photobioreactor systems were made of cylindrical glass photobioreactor with 30 cm in length and 7 cm in diameter. Each unit of photobioreactor contained 800 mL cultured microalgae. 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/hr at 2, 5, 10 and 15% CO2 aeration, respectively (Figure 3-13).

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 3-14).

Daily recovery of biomass in the six-parallel photobioreactor was determined. In each case, the amount of biomass recovered daily in the six-parallel photobioreactor was around six times greater than the amounts recovered in the single photobioreactor (Table 3-3). 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 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.

3.9. CO

utilization of Nannochloropsis oculata

3.9.1. Effect of CO₂ concentration on cell growth in semi-continuous cultures

For the study of biomass productivity in response to higher CO2 aeration, the microalgal cells pre-adapted to CO2 were applied. In the experiment, Nannochloropsis oculata cells were pre-adapted to 2% CO2 before the microalga was inoculated into the semi-continuous cultures. Moreover, a high density (approximate 0.4 g/L) of inoculum was applied in the cultures. The semi-continuous 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 Nannochloropsis oculata aerated with 2, 5, 10, and 15%

CO2 in the semi-continuous system were similar. The average specific growth rate and maximum cell density (i.e., biomass concentration) were from 0.683 to 0.733 μ and from 0.745 to 0.928 g/Lat different concentrations of CO2 aerated cultures, respectively (Figure 3-15). High CO2 aeration (5-15%) may be a harmful effect on the microalgal cells growth as shown in Figure 3-10. 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.

3.9.2. Optimal CO2 concentration applied in semi-continuous cultures

In the semi-continuous system, Nannochloropsis oculata 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 semi-continuous system cultured with

Nannochloropsis oculata 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 reduction, and CO2 removal efficiency were recorded and showed in Figure 3-16. 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/hr, 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, 2007b; 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, 2007]. 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 reduction 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 0.211, 0.234, 0.350 and 0.393 g/hr, however, total biomass productivity was 0.480, 0.441, 0.398 and 0.372 g/L/d 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 productivity was 2% CO2.

3.10. Biomass production of Nannochloropsis oculata in semi-continuous culture

In the semi-continuous culture system, the Nannochloropsis oculata cells were collected at the time before culture replaced each day for determination of biomass productivity.

Table 3-4 summarizes the biomass productivity of Nannochloropsis oculata cultures aerated with various CO2 concentrations. As increasing CO2 concentration of aeration from 2 to 15%, both biomass productivity were generally decreasing. Our results showed that the pH

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