3-1 Growth ability and lipid content of four potential strains
Microalgae strains of Chlorella sp., N. oculata, Skeletonema costatum, Isochrysis galbana and Tetraselmis chui were acquired from Taiwan Fisheries Research Institute. The cell density of these four strains was measured by optical density A682. The linear regression of cell density and optical density was established firstly for comparing growth ability in the four strains (only showed Chlorella sp. and N. oculata in this study). Figure 3-1 shows the growth ability of Chlorella sp., N. oculata, Skeletonema costatum, Isochrysis galbana and Tetraselmis chui. Chlorella sp. and N. oculata exhibited the potential for fast growth
capability. Then these four algal strains were examined on lipid content for potential of biodiesel production (Figure 3-2). As the same to the growth ability, Chlorella sp. and N.
oculata are richer in lipid content than other algal species. Nannochloropsis oculata
contained the highest lipid contents of 35% and Chlorella sp. had the secondary rich lipid contents of 17%. However, lipid contents of Skeletonema costatum, Isochrysis galbana and Tetraselmis chui are only about 9-10%.
3-2 Cultivation with CO
2and lipid composition
The potential microalgal species Chlorella sp. and N. oculata were cultured with aeration of simulated waste green-house gas CO2. The cultures were aerated with 10% CO2 for carbon source of essential photosynthesis. The growth of Chlorella sp. and N. oculata cultures with CO2 aeration was rapider than cultures only with air aeration after 3 days (Figure 3-3). However, Chlorella sp. go into stationary phase in the fourth day and N.
oculata kept growth ability.
Lipid composition of Chlorella sp. and N. oculata were analysis by Gas chromatography (Figure 3-4 and Table 3-1). The length of fatty acid chain plays an important role to decide the characteristics of biodiesel such as pour point, boiling point and so on. C12:0~C18:0 and C18:1, C18:2 and C18:3 are usual fatty acid for biodiesel contents. In lipid composition of Chlorella sp. and N. oculata, C16:0 are 32.3% and 12.0%, respectively. Therefore, the algal
oil of Chlorella sp. may have higher melting point suitable for usage in the torrid areas. In the results, the C18:1 and C18:3 could not be separated by GC in our methods. C18:2 of Chlorella sp. and N. oculata are 20.7% and 31.6%, respectively. Both C18:2 are higher than
12% that fits the definition by European legislation, but if the linolenic acids (18:3) are very abundant, the C18:3 fatty acids may require additional treatment of catalytic hydrogenation or the use in mixture with a biodiesel richer in saturated fatty acids [Converti et al., 2009].
3-3 Measurement 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, as well as optical density and cell dry weight of Chlorella sp. and N. oculata were established by linear regression firstly (Figure 3-5 and
Figure 3-6). Both cell density (R2 = 0.997; p < 0.001) and biomass (R2 = 0.991; p < 0.001) can be precisely predicted by optical density. Therefore, the values of optical density were used to calculate the related biomass of Chlorella sp. and N. oculata NCTU-3 in each experiment according the equations established in this study.
3-4 Effect of CO
2on biomass and lipid production
To investigate the effect of CO2 concentration on growth, Chlorella sp. and N. oculata in batch culture was incubated for 4-8 days at 26 ± 1℃ and 300 μmol/m2 s and aerated with different concentrations of CO2 at 0.25 vvm. The initial inoculum (approximate 8 × 105 cells/mL) was cultured and aerated with air (CO2 concentration is approximate 0.03%), 2%, 5%, 10%, and 15% CO2. 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 1-2 days batch culture in each experiment.
Figure 3-7 shows the microalgal growth of Chlorella sp. and N. oculata aerated with different CO2 concentrations. After the 6-8 days, the cells grew up to plateau stage and the biomass of Chlorella sp. in air, 2% and 5% CO2 aeration were 0.537 ± 0.016 g/L, 1.211 ± 0.031 g/L, and 0.062 ± 0.027 g/L and. Furthermore, the biomass of N. oculata in air and 2%
were 0.268 ± 0.022 and 1.277 ± 0.043 g/L, respectively. At the aeration of 2% CO2, Chlorella sp. grew most rapidly at the specific growth rate of 0.492 1/d than aeration of air
(specific growth rate is 0.230 1/d) and the specific growth rate markedly fell to be 0.127 d-1when the cultures were aerated with 5% CO2. The growth of Chlorella sp. at 10% and 15% CO2 aeration was almost completely inhibited, so the specific growth rates were not available. Similarity, the N. oculata culture aerated with 2% CO2 showed an optimal growth potential and the specific growth rate in the 2% CO2 and air aerated cultures were 0.571 1/d and 0.194 1/d. Whereas, the growth of N. oculata aerated with 5%, 10%, and 15% CO2
were completely inhibited.
When the microalgal cultures 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] and Chiu et al. [2009].
They indicated that microalga, Nannochloropsis sp., grew well 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, 5-15%, 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, 2007a, b; Chiu et al., 2009].
3-4-2 Effect of CO2 on cell growth in microalgal semicontinuous cultivation
For the study of lipid accumulation in response to higher CO2 aeration, the microalgal cells pre-adapted to CO2 were applied. In the experiment, microalgal cells were pre-adapted to 2% CO2 before the microalga was inoculated into the semicontinuous cultures.
Therefore, the semicontinuous culture was carried out in two stages. A batch culture had an initial high cell density of inoculum (approximate 8 × 106 cells/mL) firstly. At 2% CO2, cell density was allowed to increase until it reached an optical density (A682) over 5 (the cell density was about 1 × 108 cells/mL), which occurred after 6-8 days of incubation. After that, the semicontinuous system was operated and 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. These semicontinuous cultures aerated with different CO2 concentrations were operated for 24 days and the growth of microalgae was stable by each day replacement and was maintained at logarithmic growth potential. The growth of Chlorella sp. and N.
oculata in the semicontinuous culture was constantly similar at 2%, 5%, 10%, and 15% CO2. The average specific growth rate and biomass of Chlorella sp. and N. oculata, respectively, were 0.58-0.66 1/d, 0.76-0.87 g/L and 0.683-0.733 1/d, 0.745-0.928 g/L after 8 days of incubation at 2-15% CO2 aeration. N. oculata showed slight great growth potential than Chlorella sp. These results show that a high concentration of CO2 (10-15%) may directly introduce to a high-density Chlorella sp. and N. oculata culture in the semicontinuous photobioreactor system. Although, high CO2 aeration (5-15%) may be a harmful effect on
pre-adapting to 2% CO2 culture could promote the growth capacity of microalga in the cultures aerated with higher CO2 concentrations.
In the results of semicontinuous culture of microalgae, 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 cultures were adapted to 2% CO2 may overcome environment stress and drastic harmful effects on microalgal cell growth 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 [Lee et al., 2002; Yun et al., 1997]. In our
semicontinuous photobioreactors, Chlorella sp. and N. oculata 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.
3-4-3 Effect of CO2 on lipid and biomass production in semicontinuous cultures
Lipid and biomass productivity in the semicontinuous Chlorella sp. and N. oculata cultures were determined before the culture broth was replaced each day. Table 3-2 and Table 3-3 summarize the results, lipid and biomass productivity, collected from the single
replaced in the 800 mL semicontinuous photobioreactor aerated with 2, 5, 10, and 15% CO2, the total biomass productivity and lipid productivity per day (400 mL of waste broth was recovered for measurement) of Chlorella sp. in each photobioreactor was 0.421, 0.404, 0.366, and 0.361 g/L/d, and 0.143, 0.130, 0.124, and 0.121 g/L/d, respectively. Moreover, the total biomass productivity and lipid productivity per day of N. oculata culture in each
photobioreactor was 0.480, 0.441, 0.398, 0.372 g/L/d, and 0.142, 0.113, 0.097, 0.084 g/L/d, respectively. The total biomass productivity and lipid productivity of Chlorella sp. cultures were similar to N. oculata. In the result, Chlorella sp. and N. oculata are potential
candidates for biomass and lipid production by semicontinuous cultures.
However, in the single semicontinuous culture, both of lipid and biomass productivity decreased when the aerated CO2 concentration was increased (Table 3-2 and Table 3-3).
Lipid content in Chlorella sp. and N. oculata cultured at 2, 5, 10, and 15% CO2 were very similar (approximately 32-34% and 22-29% of dry weight). In the semicontinuous culture, the optimum condition for biomass productivity was at 2% CO2 aeration and lipid content was not apparently affected even by high CO2 aeration. Biomass productivity of Chlorella sp.
and N. oculata at 15% CO2 aeration were 68% and 77% of that at 2% CO2 aeration.
Nevertheless, our results still show the potential growth of microalgal Chlorella sp. and N.
oculata for lipid and biomass productivity in the semicontinuous system even the cells were cultivated in the condition aerated with 15% CO2.
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 our studies showed an inverted result may due to different microalgal species, growth condition, and medium content [Hu and Gao, 2006]. Our results show that the pH of N. oculata cultures with 2%, 5%, 10%, and 15% CO 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 microalgae may be mainly affected by pH and lipid content of the microalgal cultures was decreased with decrease of broth pH.
3-5 Lipid content of microalgae at different growth phases and nitrate limitation
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. To investigate the effect of nitrogen concentration on growth, Chlorella sp. and N. oculata in batch culture was incubated for 8-9 days at 26 ± 1℃ and 300 μmol/m2 s and aerated with 10% of CO2 at 0.25 vvm. The nitrate concentration was measured by optical density at A220. The standard curves and equations of optical density at A220 to the nitrate concentration were introduced in Figure 3-8.
Figure 3-9 and Figure 3-10 shows that the lipid accumulation in microalgal cells was associated with growth phases and nitrate concentration. The lipid content of Chlorella sp.
cells at logarithmic, early stationary phase and stationary phase was 12, 17 and 24%. And the lipid content of N. oculata NCTU-3 cells at logarithmic, early stationary phase and stationary phase was 21, 34 and 50%, respectively. This result indicated that lipid
accumulation increases as microalgae approach into stationary phase. The decreased nitrate content in the broth of microalgal culture from logarithmic phase to stationary phase was along with the growth of microalgal cells. It is hinted that the Chlorella sp. and N. oculata 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]. Report showed the knowledge that illumination of photosynthetic tissue stimulates nitrate reduction and lipid synthesis. That lipid synthesis is stimulated under nitrogen starvation indicate that photosynthetically produced reluctant may be used either for lipid synthesis or the several steps of nitrate reduction [Yung and Mudd, 1966]. The result is confirmed by these previous reports that the microalgae, Chlorella sp. and N. oculata NCTU-3, shows the metabolic effect of nitrogen depletion related to the increasing lipid accumulation.
3-6 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 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 was aerated with 2% CO2. Figure 3-11 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 days
cultivation. Table 3-4 shows the biomass and lipid productivity of N. oculata NCTU-3 cells
volume of replaced broth was 4800 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 (41.2% vs.
30.7%). 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.
3-7 Biomass and lipid production of microalgae in mixotrophic and heterotrophic cultivation with various carbon sources
3-7-1 Effects of organic carbons on the growth of N. oculata
The influence of four organic carbon sources, glucose, citric acid, sucrose and sodium acetate (10 mM), on the growth of N. oculata in mixotrophic and heterotrophic cultivations compared with photoautotrophic cultures was represented in Figure 3-12. The initial inoculum of cell density was 2 × 107 cells/mL in cultivating batch culture. N. oculata was incapable of heterotrophic growth on the supplementation of those carbon substrates (Figure 3-12). The cell densities obtained in the heterotrophic cultivations with sodium acetate, citric acid, glucose and sucrose on the fifth day were 2.0, 2.2, 2.3 and 2.5 × 107 cells/mL, respectively. Whereas, the cell density for the control culture of heterotrophic cultivations on the fifth day was only 2.9 ×107 cells/mL. The growth capabilities in all of the in heterotrophic cultivations with different carbon sources as well as the control culture of
heterotrophic cultivation (without any carbon source supplementation) were not efficient.
This indicated the growth of this N. oculata in this study did not grow well in dark condition.
The study of Vazhappilly and Chen [1998] showed N. oculata UTEX LB 2164 grew not well in heterotrophic growth when glucose and acetate was used as the sole carbon and energy source.
Compared with the cell densities of photoautotrophic culture (7.7 × 107 cells/mL), the cell densities of the cultures supplemented with sodium acetate, citric acid, glucose and sucrose on the fifth day reached 9.2, 10.4, 11.2 and 12.9 × 107 cells/mL in the mixotrophic cultivations, respectively (Figure 3-12). Overall, organic carbon supply enhanced the growth capacities of N. oculata in the mixotrophic cultivation compared with those in the heterotrophic
cultivation. The result shows that cultivation with sucrose in the mixotrophic condition obtained the best growth capacity, and the cell density was 5.2-fold higher than heterotrophic cultivation with sucrose. Sucrose gave the highest cell density; this might be the reason that the sucrose is metabolized to glucose and fructose in one equivalent each in microalgal cells and fructose might be use as the carbon source. Glucose and fructose were used as organic carbon substrates in the cultures of microalgae such as a blue green alga, cyanobacterium Anabaena variabilis [Pearce and Carr 1969; Valiente et al. 1992].
However, the growth capability of mixotrophic culture with acetate and citrate was lower than the other organic carbon source. The same as our result, wood et al. [1999] indicated that the Nannochloropsis strains seemed to be particularly sensitive to acetate inhibition.
However, some researches showed Nannochloropsis sp. can cultivate in mixotrophic culture with supply of acetate but it cannot uptake glucose [Hu and Gao 2003]. And in
Nannochloropsis strain investigated by Liang et al. [2009], acetate utilization especially when nitrogen was limited, provided higher lipid content compared with those from growth on glucose. Consequently, various organic carbon sources may make different effects in
different species even in Nannochloropsis strains.
3-7-2 Effects of inorganic and organic carbons on the growth of N. oculata
In Figure 3-13, the photoautotrophic culture of N. oculata cultured with 2% aeration of CO2 is slightly slower than the culture with the source of sucrose (11.1 × 107 cells/mL vs. 12.9
× 107 cells/mL), and approximately same as the culture with glucose (11.2 ×107 cells ml-1), but higher than those with the supplementation of sodium acetate and citric acid in
mixotrophic condition and the control culture of mixotrophic cultivations. Carbon sources are necessary to provide the carbon skeletons and energy for cell growth [Wen and Chen 2003]. In this study, cultivation of N. oculata using sucrose as carbon substrate in
mixotrophic growth gave the highest growth capability. The synergistic effect of light and the organic carbon is essential for high ATP production and biomass productivity in
mixotrophic culture [Cid et al., 1992; Yang et al., 2000]. Mixotrophic growth doesn’t require high light intensities, and consequently could reduce the energy costs of microalgal cultures and photobioreactors [Read et al., 1989; Fernández Sevilla et al., 2004].
3-7-3 Lipid contents and production in N. oculata cultures with different carbon sources
Lipid production of N. oculata cultures in mixotrophic and heterotrophic cultivations as well as the control cultures in those two cultivations were illustrated in Figure 3-14. Lipid contents of the mixotrophic cultivations supplemented with sodium acetate, citric acid, glucose and sucrose were 39, 37, 37 and 36%, respectively. Whereas, the lipid contents of the heterotrophic cultivations with sucrose, citric acid, sodium acetate and glucose were 54, 29, 25 and 20%. Besides, lipid contents produced in the control culture of mixotrophic cultivations was only 13% and in the control culture of heterotrophic cultivations was only 11%. Among these mixotrophic and heterotrophic growths, the cultivation with sucrose provided the highest lipid content in the heterotrophic cultivation. This suggested that the
energy produced in the cultivation with sucrose in heterotrophic growth was used for the production of lipid compounds. The report of Xu et al. [2004] also showed the heterotrophic Nannochloropsis sp. growth gave the highest production of lipid (42.7%) compared to
mixotrophic growth (38.7%) and photoautotrophic (38.1%).
With the supplement of the organic carbon sources in mixotrophic condition, the biomass of the cultures supplemented with sodium acetate, citric acid, glucose and sucrose were 0.538 and 0.641, 0.690 and 0.798 g/L, whereas lipid production in the cultures supplemented with
With the supplement of the organic carbon sources in mixotrophic condition, the biomass of the cultures supplemented with sodium acetate, citric acid, glucose and sucrose were 0.538 and 0.641, 0.690 and 0.798 g/L, whereas lipid production in the cultures supplemented with