Lipid accumulation and CO
2
utilization of Nannochloropsis oculata
in response to CO
2
aeration
Sheng-Yi Chiu
a, Chien-Ya Kao
a, Ming-Ta Tsai
a, Seow-Chin Ong
a, Chiun-Hsun Chen
b, Chih-Sheng Lin
a,*a
Department of Biological Science and Technology, National Chiao Tung University, No. 75 Po-Ai Street, Hsinchu 30068, Taiwan
bDepartment of Mechanical Engineering, National Chiao Tung University, Hsinchu 30068, Taiwan
a r t i c l e
i n f o
Article history: Received 29 March 2008
Received in revised form 26 June 2008 Accepted 27 June 2008
Available online 22 August 2008 Keywords: Nannochloropsis oculata Lipid Biomass Carbon dioxide
a b s t r a c t
In order to produce microalgal lipids that can be transformed to biodiesel fuel, effects of concentration of CO2aeration on the biomass production and lipid accumulation of Nannochloropsis oculata in a semicon-tinuous culture were investigated in this study. Lipid content of N. oculata cells at different growth phases was also explored. The results showed that the lipid accumulation from logarithmic phase to stationary phase of N. oculata NCTU-3 was significantly increased from 30.8% to 50.4%. In the microalgal cultures aerated with 2%, 5%, 10% and 15% CO2, the maximal biomass and lipid productivity in the semicontinuous system were 0.480 and 0.142 g L1d1with 2% CO
2aeration, respectively. Even the N. oculata NCTU-3 cultured in the semicontinuous system aerated with 15% CO2, the biomass and lipid productivity could reach to 0.372 and 0.084 g L1d1, respectively. In the comparison of productive efficiencies, the semi-continuous system was operated with two culture approaches over 12 d. The biomass and lipid produc-tivity of N. oculata NCTU-3 were 0.497 and 0.151 g L1d1in one-day replacement (half broth was replaced each day), and were 0.296 and 0.121 g L1d1in three-day replacement (three fifth broth was replaced every 3 d), respectively. To optimize the condition for long-term biomass and lipid yield from N. oculata NCTU-3, this microalga was suggested to grow in the semicontinuous system aerated with 2% CO2and operated by one-day replacement.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Greenhouse gases are accumulating dramatically in Earth’s atmosphere as a result of human activities and industrialization. In addition, the increasing concentration of greenhouse gases causes serious global warming increasing the temperatures of the surface air and subsurface ocean. Carbon dioxide (CO2) is the main
greenhouse gas. Many attempts including physical and chemical treatments have been used to recover CO2 from atmosphere. In
biological approach, microalgae appear more photosynthetically efficient than terrestrial plants and are the candidates as efficient CO2fixers (Brown and Zeiler, 1993).
In recent years, the bioregenerative methods using photosyn-thesis by microalgal cells have been made to reduce the atmo-spheric CO2to ensure a safe and reliable living environment. As
the result of mild conditions for CO2fixation, there is no
require-ment for further disposal of recovered CO2 (Lee and Lee, 2003;
Cheng et al., 2006; Jin et al., 2006). Marine microalgae are expected as a proper candidate due to their high capability for photosynthe-sis and easily cultured in sea water which solubilizes high amount
of CO2. The CO2fixation by microalgal photosynthesis and biomass
conversion into liquid fuel is considered a simple and appropriate process for CO2circulation on Earth (Takagi et al., 2000).
Lipids from microalgae are chemically similar to common veg-etable oils and have been suggested being a high potential source of biodiesel (Dunahay et al., 1996; Chisti, 2007). Microalgal oil most accumulated as triglycerides can be transformed to biodiesel (Lee et al., 1998; Zhang et al., 2003). The biodiesel compared with fossil-driven diesel, that is renewable, biodegradable, and low pol-lutant produced (Vicente et al., 2004). The advantages of biodiesel from microalgae are that microalgae are easy to culture and less area occupation for cultivation (Chisti, 2007). In addition, microal-gal-based biodiesel is a potential renewable resource for displace-ment liquid transport fuels derived from petroleum (Chisti, 2008). Nannochloropsis oculata is an interesting microorganism in the field of marine biotechnology because of its high lipid content. Many microalgae can accumulate lipids due to excess photosyn-thate and some species can accumulate amount of lipids under het-erotroph or environment stress, such as nutrient deficiency (Takagi et al., 2000) or salt stress (Takagi et al., 2006). In this study, we investigated the effects of CO2concentration in airstreams on the
biomass production and lipid accumulation of N. oculata NCTU-3 cultures. We also evaluated the efficiency of lipid productivity in
0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.06.061
* Corresponding author. Tel.: +886 3 5131338; fax: +886 3 5729288. E-mail address:lincs@mail.nctu.edu.tw(C.-S. Lin).
Contents lists available atScienceDirect
Bioresource Technology
a semicontinuous system for N. oculata NCTU-3 cultures. For a long-term operation, the total biomass and lipid productivity in the semicontinuous system operated by one-day and three-day replacement were evaluated.
2. Methods
2.1. Microalgal cultures, medium, and chemicals
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 bio-mass production at National Chiao Tung University, Taiwan (data not shown). The N. oculata NCTU-3 cells were grown in the modi-fied f/2 medium in artificial sea water which has the following composition (per liter): 29.23 g NaCl, 1.105 g KCl, 11.09 g MgSO47H2O, 1.21 g tris-base, 1.83 g CaCl2.2H2O, 0.25 g NaHCO3,
and 3.0 mL of trace elemental solution. The trace elemental solu-tion (per liter) includes 75 g NaNO3, 5 g NaH2PO4H2O, 4.36 g
Na2EDTA, 3.16 g FeCl36H2O, 180 mg MnCl24H2O, 10 mg
CoCl26H2O, 10 mg CuSO45H2O, 23 mg ZnSO47H2O, 6 mg Na
2-MoO4, 100 mg vitamin B1, 0.5 mg vitamin B12and 0.5 mg biotin.
2.2. Preparation of microalgal inoculum
A stock culture of N. oculata NCTU-3 (approximately 1 105cells mL1) was cultured in an Erlenmeyer flask with
800 mL working volume of modified f/2 medium under 26 ± 1 °C and 300
l
mol m2s1. After 6 d culture, the microalgal cellspel-leted by centrifugation at 3000 g for 5 min were resuspended with 50 mL fresh medium and separated for further experiments. Light intensity was measured from the light-attached surface of the photobioreactor using a Basic Quantum Meter (Spectrum Tech-nologies, Inc., Plainfield, IL, USA).
2.3. Experimental system with photobioreactor
The N. oculata NCTU-3 was cultured in a cylindrical glass photo-bioreactor (30 cm length, 7 cm diameter) with 800 mL of working volume placed at 26 ± 1 °C under continuous, cool white, fluores-cent lights. The setup of photobioreactor for microalgal culture sys-tem was described in the previous research (Chiu et al., 2008). Light intensity was approximately 300
l
mol m2s1at the surfaceof photobioreactor. Gas provided as different concentrations of CO2
mixed with ambient air was prepared with a volumetric percent-age of CO2 and filtered (0.22
l
m) to give CO2 concentrations of2%, 5%, 10%, or 15%. The microalgal cultures were aerated continu-ously with gas provided via bubbling from the bottom of reactor with an aeration rate of 200 mL min1(i.e., 0.25 vvm, volume gas
per volume broth per min). A pre-cultured N. oculata NCTU-3 was inoculated in cylindrical glass photobioreactor in 800 mL cul-ture volume at an initial biomass concentration (calculated dried weight of microalgal cells per liter, g L1) of 0.01 g L1
(approxi-mate 7 105cells mL1) as a batch culture. Different
concentra-tions of CO2 aeration were mixed with air and pure CO2, and
adjusted by gas flow meter (Dwyer Instruments, Inc., Michigan, IN, USA) to give a flow rate of 0.25 vvm.
2.4. Microalgal cell counting and dry weight
A direct microscopic count was performed with Brightline Hemocytometer (BOECO, Hamburg, Germany) and a Nikon Eclipse TS100 inverted metallurgical microscope (Nikon Corporation, To-kyo, Japan). Cell density (cells mL1) was measured by an Ultrospec
3300 pro UV/Visible spectrophotometer (Amersham Biosciences,
Cambridge, UK) at the absorbance of 682 nm (A682). Each sample
was diluted to give an absorbance in the range 0.1–1.0 if optical density was greater than 1.0.
Microalgal dry weight per liter (g L1) was measured according
to the method previously reported (American Public Health Associ-ation, 1998). Microalgal cells were collected by centrifugation and washed twice with deionized water. Microalgal pellet was dried at 105 °C for 16 h for dry weight measurement (Takagi et al., 2006). 2.5. Measurement of growth rate
A regression equation of the cell density and dry weight per liter of culture was obtained by a spectrophotometric method (Chiu et al., 2008). Specific growth rate (
l
d1) was calculated as follows:l
¼lnðWf=WoÞD
twhere Wfand W0were the final and initial biomass concentration,
respectively. 4t was the cultivation time in day (Ono and Cuello, 2007).
2.6. Chemical analyses
Sample pH was directly determined with an ISFET pH meter KS723 (Shindengen Electric Mfg Co. Ltd., Tokyo, Japan).
Determination of nitrate content in broth was followed by the spectrometric method reported byCollos et al. (1999). Broth from microalgal cultures was collected and centrifuged at 3000 g for 5 min. The absorbance of supernatant was measured at 220 nm. A standard curve was determined from authentic sodium nitrate at concentrations from 0 to 440
l
M.The CO2 concentration in airstreams, CO2(g), was measured
using a Guardian Plus Infra-Red CO2Monitor D-500 (Edinburgh
Instruments Ltd, Livingston, UK).
Lipid extraction was according to the modified method reported byTakagi et al. (2006). The microalgal cells were obtained by cen-trifugation at 3000 g for 15 min. Cells were washed with deion-ized water twice, and the dried biomass was obtained by lyophilization. A sample (30 mg) was mixed with methanol/chlo-roform solution (2:1, v/v) and sonicated for 1 h. After precipitation of mixture with methanol/chloroform solution, chloroform and 1% NaCl solution were then added to give a ratio of methanol, chloro-form, and water of 2:2:1. The mixture was centrifuged at 1000 g for 10 min and the chloroform phase was recovered. Finally, chlo-roform was removed under vacuum in a rotary evaporator and the remainder was weighed as lipid.
2.7. Measurement of lipid content by fluorescent spectrometry For fast determination of lipid content, a fluorescent spectro-metric method was applied. In the method, the microalgal cells were stained with Nile Red (Sigma, St. Louis, MO, USA) followed the protocol reported byde la Jara et al. (2003). In brief, 1 mL of 1 106cells suspension was added 50
l
L of Nile Red in acetoneworking solution as a concentration of 0.1 mg mL1for lipid
stain-ing. 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 were used. Non-stained cells were used as an auto-florescence control. The rel-ative 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.
2.8. Setup of semicontinuous culture system
Before the N. oculata NCTU-3 cultures applied to the semicon-tinuous system aerated with various CO2 concentrations, the
microalga was grown in a batch culture and aerated with air. When the cell density in the batch culture reached to about 1 107cells mL1, the culture was changed into the aeration of
2% CO2. After 4–6 d cultivation aerated with 2% CO2, cell density
of the culture reached up to about 1 108cells mL1. The culture
was then replaced half of broth with fresh medium each day and performed for 3 d. After that, the culture was also replaced half of broth with fresh medium at the forth day and aerated with 2, 5, 10, and 15% CO2. After 4 d culture, the sampling time was at 6,
12, and 24 h everyday and the culture was replaced half of broth with fresh medium daily.
2.9. 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.
3. Results and discussion
3.1. Growth of N. oculata NCTU-3 aerated with different CO2
concentration
Effect of CO2concentration in airstream on the growth of N.
ocu-lata NCTU-3 was investigated in a batch culture incubated at 26 ± 1 °C and 300
l
mol m2s1. The initial biomass inoculumwas 0.01 g L1 (about 7 105cells mL1) 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
inter-val. The specific growth rate was calculated from the cultures in each experiment.Fig. 1shows the microalgal growth aerated with different CO2concentrations. After 6–8 d, the growth of air and 2%
CO2aerated 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 L1, 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 d1 and 0.571 d1, 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 en-hanced compared with those in the culture aerated with air. This result was confirmed byHu and Gao (2003). They indicated that microalga, Nannochloropsis sp., grew best in an enriched CO2
aera-tion compared with air aeraaera-tion. 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 CO2aeration
above 5% could be harmful to microalgal cells and inhibit the mic-roalgal growth (Silva and Pirt, 1984; Sung et al., 1999; Chang and Yang, 2003; de Morais and Costa, 2007a,b).
3.2. 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 log-arithmic, early stationary phase and stationary phase were 30.8, 39.7, and 50.4%, respectively. This result indicated that lipid accu-mulation increases as N. oculata NCTU-3 approaches into station-ary 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 accom-pany with the nitrate depletion. Roessler (1988) reported that the nutrient deficiency induced an increase in the rate of lipid syn-thesis in a diatom, Cyclotella cryptica, and resulted in lipid accumu-lation in the cells. It is also indicated that lipid accumuaccumu-lation 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.
3.3. Effect of CO2concentration on cell growth in semicontinuous
cultures
For the study of lipid accumulation in response to higher CO2
aeration, the microalgal cells pre-adapted to CO2were applied. In
the experiment, N. oculata NCTU-3 cells were pre-adapted to 2% CO2before the microalga was inoculated into the semicontinuous
cultures. Moreover, a high density (approximate 0.4 g L1) of
inoc-ulum was applied in the cultures. The semicontinuous system was operated for 8 d and the growth was stable by each day replace-ment and was maintained at logarithmic growth potential. The re-sults showed that the growth profiles of N. oculata NCTU-3 aerated with 2%, 5%, 10%, and 15% CO2in the semicontinuous system were
similar (Fig. 2). The average specific growth rate and maximum cell density (i.e., biomass concentration) were from 0.683 to 0.733 d1
and from 0.745 to 0.928 g L1at different concentrations of CO 2
aerated cultures, respectively (Fig. 2). High CO2aeration (5–15%)
may be a harmful effect on the microalgal cells growth as shown inFig. 1. But increasing the inoculated cell density and pre-adapt-ing to 2% CO2culture could promote the growth capacity of
micro-alga in the cultures aerated with higher CO2concentrations. The
results indicated that increasing cell density and pre-adapting mic-roalgal cells in an adequate CO2concentration is an alternative
ap-proach for the application of high CO2 aeration without drastic
harmful effects on microalgal cell growth.
1.6 0.8 1.2 15% 10% 5% 2% Air 0 0.4 0 Biomass concentration (g L -1) Days of cultivation 1 2 3 4 5 6 7 8
Fig. 1. Effect of the concentrations of CO2aeration on the growth of N. oculata
NCTU-3. In the cultures, approximate 0.01 g L1
of microalgal cells was inoculated and cultivated under air, 2%, 5%, 10%, and 15% CO2aeration. All experiments were
carried out in triplicate. The cultures were illuminated at 300lmol m2s1and
3.4. 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 1 sum-marizes the biomass and lipid productivity of N. oculata NCTU-3 cultures aerated with various CO2 concentrations. As increasing
CO2concentration of aeration from 2 to 15%, both biomass and
li-pid productivity were generally decreasing (Table 1). It is reported that the lipid content was increasing associated with the increasing CO2concentration 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 microalgal species, growth condition, and medium content (Hu and Gao, 2006). Our results showed 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 synthe-sis was decreased with decrease of pH. This may be possibly be-cause 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 af-fected by pH and lipid content of the microalgal cultures was de-creased with decrease of broth pH.
3.5. Optimal CO2concentration 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 CO2removal. Therefore, the CO2
re-moval 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 CO2between influent and effluent, and CO2
re-moval efficiency were recorded. The CO2 concentrations in the
effluent of 2, 5, 10 and 15% CO2aerated cultures were maintained
at 0.9–1.1, 3.8–4.1, 8.3–8.7, and 12.9–13.2% CO2over 8 d
cultiva-tion, respectively. The CO2removal efficiency in the cultures
aer-ated with 2%, 5%, 10%, and 15% CO2were 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 h1, respectively. The efficiency of CO 2
re-moval in the culture aerated with low CO2concentration was
high-er than those ahigh-erated with high CO2concentration (de Morais and
Costa, 2007a,b; Chiu et al., 2008). The CO2removal efficiency in a
closed photobioreactor system is dependent on microalgal species, photobioreactor, and concentration of CO2aeration (Cheng et al.,
2006; de Morais and Costa, 2007a,b). This assumption was con-firmed by the study in Chlorella sp., the study showed more CO2
re-moval capacity but lower biomass productivity in a microalgal culture treated with low CO2aeration (Chiu et al., 2008). Cheng
et al. (2006) demonstrated a Chlorella vulgaris cultured mem-brane-photobioreactor obtained a maximum rate of microalgal CO2fixation at 2% of CO2aeration. 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 h1. However, total biomass productivity was 0.480,
0.441, 0.398, and 0.372 g L1d1in the cultures with 2%, 5%, 10%,
and 15% CO2aeration, respectively. The microalgal cultures aerated
with higher CO2showed lower biomass productivity. This result
may due to that when the microalgal cells aerated with higher CO2, most of the CO2is consumed for metabolic activity and less
of CO2is fixed to become cellular components, i.e., biomass. The
higher metabolic activity may contribute to the microalgal cells to subsist on higher CO2stress. The results showed that the
maxi-mal CO2utilization efficiency was from the cultures aerated with
2% CO2airstreams. It is also indicated that the optimal
concentra-tion of CO2aeration in the system based on the efficiency of
bio-mass and lipid productivity was 2% CO2.
3.6. Comparison of productive efficiencies in semicontinuous system with different culture approaches
The comparison of productive efficiencies in the semicontinu-ous systems in which the culture broth were replaced at an inter-val of 24 h (one-day replacement) or 72 h (three-day replacement)
Biomass concentration (g L -1) 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1 0 1 2 3 4 5 6 7 8 Days of cultivation 2% 5% 10% 15% 1.2 1.2 1.2 1.2
Fig. 2. Growth profiles of N. oculata NCTU-3 cultured in the semicontinuous system aerated with 2%, 5%, 10%, and 15% CO2. In the cultures, approximate 0.4 g L1of
microalgal cells was inoculated and cultivated under an illumination at 300lmol m2
s1
and bubbled with a flow rate of 0.25 vvm airstreams at 26 ± 1 °C for 8 d. Amount of 50% of cultured broth was replaced with the fresh modified f/2 medium at interval of 24 h.
Table 1
Daily recovery of biomass and lipid of N. oculata NCTU-3 cultured in the semicon-tinuous system aerated with different CO2concentrations
CO2
aeration
Total biomass productivity (cell dry weight, g L1d1)
Total lipid productivity (g L1d1) Percentage of lipid content (%) 2% 0.480 ± 0.029 0.142 ± 0.049 29.7 ± 2.0 5% 0.441 ± 0.044 0.113 ± 0.035 26.2 ± 1.9 10% 0.398 ± 0.069 0.097 ± 0.026 24.6 ± 1.7 15% 0.372 ± 0.022 0.084 ± 0.021 22.7 ± 1.9 The semicontinuous cultures were performed for 8 d and a half of broth was replaced each day. The culture volume in photobioreactor is 800 mL. Daily waste broth was 400 mL. Each data indicates the mean ± SD, which were measured daily from d-1 to d-8.
was performed. In the systems, approximate 0.4 g L1of N. oculata
NCTU-3 cells was inoculated and the microalgal cultures were re-placed 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.Fig. 3shows
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 re-placed before the cells reached to early stationary phase in day replacement. The growth profiles of both one-day and three-day replacement cultures were stable over 12 d cultivation.Table 2shows 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 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 bio-mass 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 be-cause of nutrition-deficient effect.
4. Conclusions
The results showed the lipid accumulation of N. oculata NCTU-3 could be increased from logarithmic growth phase to stationary growth phase. The N. oculata NCTU-3 pre-adapting to 2% CO2
cul-tured in a semicontinuous system with a high cell density of inoc-ulum could grow well in the system aerated with higher CO2
concentration (5–15% CO2); however, increasing biomass
produc-tion and lipid accumulaproduc-tion would not be followed as the cultures aerated with higher CO2. Achieving the optimal condition for a
long-term biomass and lipid yield in the semicontinuous system, the microalga could be cultured with 2% CO2aeration in one-day
replacement operation.
Acknowledgements
The work was financially supported by the Research Grants of NSC 94-2218-E-009-032, NSC 95-2218-E-009-018 and NSC 96-2218-E-009-002 from National Science Council, Taiwan. This work was also partially supported by the Ministry of Education, under Grant of MoE ATU Program, Taiwan.
References
American Public Health Association, 1998. Methods for biomass production. In: Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Baltimore, MD, USA.
Brown, L.M., Zeiler, K.G., 1993. Aquatic biomass and carbon dioxide trapping. Energy Conv. Manag. 34, 1005–1013.
Carvalho, A.P., Meireles, L.A., Malcata, F.X., 2006. Microalgae reactors: a review of enclosed system designs and performances. Biotechnol. Prog. 22, 1490–1506. Chang, E.H., Yang, S.S., 2003. Some characteristics of microalgae isolated in Taiwan
for biofixation of carbon dioxide. Bot. Bul. Acad. Sin. 44, 43–52.
Cheng, L., Zhang, L., Chen, H., Gao, C., 2006. Carbon dioxide removal from air by microalgae cultured in a membrane-photobioreactor. Sep. Purif. Technol. 50, 324–329.
Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26,
126–131.
Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008. Reduction of CO2
by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99, 3389–3396.
Collos, Y., Mornet, F., Sciandra, A., Waser, N., Larson, A., Harrison, P.J., 1999. An optical method for the rapid measurement of micromolar levels of nitrate in marine phytoplankton cultures. J. Appl. Phycol. 11, 179–184.
de la Jara, A., Mendoza, H., Martel, A., Molina, C., Nordströn, L., de la Rosa, V., Díaz, R., 2003. Flow cytometric determination of lipid content in a marine dinoflagellate, Crypthecodinium cohnii. J. Appl. Phycol. 15, 433–438.
de Morais, M.G., Costa, J.A.V., 2007a. Biofixation of carbon dioxide by Spirulina sp and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor. J. Biotechnol. 129, 439–445.
(A)
One-day replacement1 1.2 0.6 0.8 1 0.2 0.4 0 6 10 12 Days of cultivation
(B)
Three-day replacement 1.6 0.8 1 1.2 1.4 0.2 0.4 0.6 0 10 12 Days of cultivation 2 4 8 2 4 6 8 Biomass concentration (g L ) Biomass concentration (g L ) -1 -1Fig. 3. Growth profiles of N. oculata NCTU-3 cultured in the semicontinuous system with 2% CO2aeration and operated by one-day and three-day replacements. In the
cultures, approximate 0.4 g L1of microalgal cells was inoculated and cultivated
under an illumination at 300lmol m2
s1
and bubbled with a flow rate of 0.25 vvm airstreams at 26 ± 1 °C. The cultivations were continuously operated for 12 d. Amount of half and three fifth of cultured broth was replaced with the fresh modified f/2 medium at interval of 24 h (one-day replacement; half broth was replaced each day) and 72 h (three-day replacement; three fifth broth was replaced every 3 d), respectively. The arrows indicate the time when the cultured broth was removed and fresh medium was added.
Table 2
Biomass and lipid productivity of N. oculata NCTU-3 cultured in the semicontinuous system aerated with 2% CO2 under the treatments of one-day and three-day
replacement
Culture Total biomass productivity (cell dry weight, g L1 d1 ) Total lipid productivity (g L1d1) Percentage of lipid content (%) One-day replacement 0.497 ± 0.032 0.151 ± 0.021 30.7 ± 2.4 Three-day replacement 0.296 ± 0.009 0.121 ± 0.035 41.2 ± 1.9
The semicontinuous cultures were performed for 12 d. The cultural broth was replaced by half (for one-day replacement) or three fifth (for three-day replace-ment) with fresh medium at interval of 24 and 72 h, respectively. The total biomass and lipid productivity were measured from the total replaced broth divided by day. The total replaced broth volume was 4800 mL in one-day replacement and only 1920 mL in three-day replacement over 12 d. Each data indicates the mean ± SD.
de Morais, M.G., Costa, J.A.V., 2007b. Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide. Energy Conv. Manag. 48, 2169–2173.
Dickson, L.G., Galloway, R.A., Patterson, G.W., 1969. Environmentally-induced changes in the fatty acids of Chlorella. Plant Physiol. 44, 1413–1416. Dunahay, T.G., Jarvis, E.E., Dais, S.S., Roessler, P.G., 1996. Manipulation of microalgae
lipid production using genetic engineering. Appl. Biochem. Biotechnol., 223– 231.
Hu, H., Gao, K., 2003. Optimization of growth and fatty acid composition of a unicellular marine picoplankton, Nannochloropsis sp., with enriched carbon sources. Biotechnol. Lett. 25, 421–425.
Hu, H., Gao, K., 2006. Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2
concentration. Biotechnol. Lett. 28, 987–992.
Jin, H.F., Lim, B.R., Lee, K., 2006. Influence of nitrate feeding on carbon dioxide fixation by microalgae. J. Environ. Sci. Health Part A-Toxic/Hazard. Subst. Environ. Eng. 41, 2813–2824.
Lee, J.S., Lee, J.P., 2003. Review of advances in biological CO2mitigation technology.
Biotechnol. Bioprocess Eng. 8, 354–359.
Lee, S.J., Yoon, B.D., Oh, H.M., 1998. Rapid method for the determination of lipid from the green alga Botryococcus braunii. Biotechnol. Tech. 12, 553–556.
Liu, Z.Y., Wang, G.C., Zhou, B.C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99, 4717– 4722.
Ono, E., Cuello, J.L., 2007. Carbon dioxide mitigation using thermophilic cyanobacteria. Biosyst. Eng. 96, 129–134.
Roessler, P.G., 1988. Changes in the activities of various lipid and carbohydrate biosynthetic enzymes in the diatom Cyclotella cryptica in response to silicon deficiency. Arch. Biochem. Biophys. 267, 521–528.
Roessler, P.G., Bleibaum, J.L., Thompson, G.A., Ohlrogge, J.B., 1994. Characteristics of the gene that encodes acetyl-CoA carboxylase in the diatom Cyclotella cryptica. Ann. N. Y. Acad. Sci. 721, 250–256.
Silva, H.J., Pirt, S.J., 1984. Carbon dioxide inhibition of photosynthetic growth of Chlorella. J. Gen. Microbiol. 130, 2833–2838.
Sung, K.D., Lee, J.S., Shin, C.S., Park, S.C., Choi, M.J., 1999. CO2fixation by Chlorella sp.
KR-1 and its cultural characteristics. Bioresour. Technol. 68, 269–273. Takagi, M., Watanabe, K., Yamaberi, K., Yoshida, T., 2000. Limited feeding of
potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp UTEX LB1999. Appl. Microbiol. Biotechnol. 54, 112–117. Takagi, M., Karseno, Yoshida, T., 2006. Effect of salt concentration on intracellular
accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J. Biosci. Bioeng. 101, 223–226.
Vicente, G., Martínez, M., Aracil, J., 2004. Integrated biodiesel production: a comparison of different homogeneous catalysts systems. Bioresour. Technol 92, 297–305.
Yongmanitchai, W., Ward, O.P., 1991. Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions. Appl. Environ. Microbiol. 57, 419–425.
Yung, K.H., Mudd, J.B., 1966. Lipid synthesis in the presence of nitrogenous compounds in Chlorella pyrenoidosa. Plant Physiol. 41, 506–509.
Zhang, Y., Dubé, M.A., McLean, D.D., Kates, M., 2003. Biodiesel production from waste cooking oil. 1. Process design and technological assessment. Bioresour. Technol. 89, 1–16.
