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Journal of Biotechnology 129 (2007) 439445

Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus

obliquus cultivated in a three-stage serial tubular photobioreactor
Michele Greque de Morais, Jorge Alberto Vieira Costa
Laboratory of Biochemical Engineering, Department of Chemistry, Federal University Foundation of Rio Grande,
P.O. Box 474, Rio Grande 96201-900, RS, Brazil
Received 28 August 2006; received in revised form 4 January 2007; accepted 12 January 2007

The increase in the concentration of atmospheric carbon dioxide is considered to be one of the main causes of global warming. As estimated
by the Intergovernmental Panel on Climate Change (IPCC) criteria, about 1015% of the gases emitted from the combustion coal being in the
form of carbon dioxide. Microalgae and cyanobacteria can contribute to the reduction of atmospheric carbon dioxide by using this gas as carbon
source. We cultivated the Scenedesmus obliquus and Spirulina sp. at 30 C in a temperature-controlled three-stage serial tubular photobioreactor
and determined the resistance of these organisms to limitation and excess of carbon dioxide and the capacity of the system to fix this greenhouse
gas. After 5 days of cultivation under conditions of carbon limitation both organisms showed cell death. Spirulina sp. presenting better results for
all parameters than S. obliquus. For Spirulina sp. the maximum specific growth rate and maximum productivity was 0.44 d1 , 0.22 g L1 d1 , both
with 6% (v/v) carbon dioxide and maximum cellular concentration was 3.50 g L1 with 12% (v/v) carbon dioxide. Maximum daily carbon dioxide
biofixation was 53.29% for 6% (v/v) carbon dioxide and 45.61% for 12% carbon dioxide to Spirulina sp. corresponding values for S. obliquus
being 28.08% for 6% (v/v) carbon dioxide and 13.56% for 12% (v/v) carbon dioxide. The highest mean carbon dioxide fixation rates value was
37.9% to Spirulina sp. in the 6% carbon dioxide runs.
2007 Elsevier B.V. All rights reserved.

Keywords: Carbon dioxide fixation; Chlorophyta; Cyanobacteria; Global warming; Greenhouse gases; Microalgae

1. Introduction dioxide, the principal greenhouse gas. According to the French

National Center for Scientific Research (CNRS), the level
The growth in human population has stimulated the search of atmospheric carbon dioxide has historically been between
for alternative food sources and new ecological technologies. 180 and 260 ppm but during the last 100 years the atmo-
Microalgae and cyanobacteria use solar light as their main spheric concentration of this gas has risen to between 260 and
source of energy, possess the potential for high productivity, are 380 ppm (Siegenthaler et al., 2005), mainly due to burning fossil-
tolerant to alterations in environmental conditions and they can fuels associated with increased population and industrialization
be cultivated in areas which are unstable for agriculture (Costa (Chang and Yang, 2003). Coupling the cultivation of photosyn-
et al., 2000). The cultivation conditions of these organisms can thetic microorganisms with the biofixation of carbon dioxide has
be manipulated to induce the production of proteins, fatty-acids, the potential not only to reduce the costs of culture media for
vitamin A, minerals, pigments and other bio-compounds and growing such organisms on an industrial scale but also to offset
their biomass can be used as a dietary supplement for humans carbon emissions (Beneman and Hughes, 1997).
and animals, including for aquaculture (Ono and Cuello, Photosynthetic microorganisms are normally cultivated in
2004). open raceway tanks using natural or artificial light but this
Photosynthetic microorganisms use inorganic carbon for requires large cultivation areas and suffers from various
growth and hence can be used for the biofixation of carbon disadvantages, including difficulty in controlling cultivation
conditions (Costa et al., 2006), evaporation of the cultivation
medium and reduced light intensity with increased depth. An
Corresponding author. Tel.: +55 53 3233 8653; fax: +55 53 3233 8745. alternative is the use of tubular photobioreactors, but Luo et al.
E-mail address: (J.A.V. Costa). (2003) have pointed out that the configuration of such reactors

0168-1656/$ see front matter 2007 Elsevier B.V. All rights reserved.
440 M.G. de Morais, J.A.V. Costa / Journal of Biotechnology 129 (2007) 439445

is one of the main factors controlling the biomass productivity and a sintered sparger, the effluent air (with or without car-
of photosynthetic cultures grown under these conditions. bon dioxide, see below) from CPBR1 being fed to the sparger
Appropriately designed photobioreactors can reduce the cul- in CPBR2 and the effluent from this photobioreactor being
tivation area by distributing photosynthetic organisms vertically, fed to CPBR3. The photobioreactors were maintaining in a
vertical tubular photobioreactors also increasing the carbon 30 C growth chamber under a 12 h dark/light photoperiod with
dioxide residence time in the cultivation medium and, con- 3200 lx of illumination provided by 40 W daylight-type fluo-
sequently, the carbon dioxide utilization efficiency (Ono and rescent lamps (General Electric, Brazil) during the light period
Cuello, 2004). Cultivations can also be carried out in serial pho- (Reichert et al., 2006).
tobioreactors in which unused effluent carbon dioxide from one For experiments without carbon dioxide filter-sterilized air
photobioreactor is fed into another photobioreactor. was bubbled through 1N sodium hydroxide and then twice
The objective of the work described in this paper was to culti- through water to remove the approximately 0.038% (v/v) present
vate the photosynthetic microorganisms Scenedesmus obliquus in air. In the experiments with carbon dioxide filter-sterilized
and Spirulina sp. in serial tubular photobioreactors and deter- carbon dioxide (White Martins, Brazil) was added to the air
mine carbon dioxide fixation and their resistance to carbon entering CPBR1 at a rate of 0.3 vvm for 15 min every 2 h during
dioxide limitation and excess. the 12 h light period such that the final carbon dioxide concen-
tration in the media was 6% or 12% (v/v). The initial biomass
2. Material and methods concentration in all runs was 0.15 g L1 and all runs lasted for
21 days.
2.1. Microorganisms and cultivation media
2.3. Analytic determinations
The Spirulina sp. (Cyanobacteria, Oscillatoriales) and S.
obliquus (Chlorophyta, Chlorophyceae) (de Morais and Costa, Triplicate samples of culture media were collected asepti-
2007) were from stock cultures kept in our laboratory. We cally at 24 h intervals and pH determined with a Q400H digital
used carbon-free media for the maintenance and cultivation pH meter (Quimis, Brazil) and biomass concentration (X, g L1 )
of both organisms, modified Zarrouk medium (de Morais and calculated by measuring the optic density at 670 nm (Costa et
Costa, 2007; Zarrouk, 1966) for Spirulina sp. and MC medium al., 2002) using a 700 Plus spectrophotometer (Femto, Brazil)
(Watanabe, 1960) for S. obliquus. For the experiments, inocu- and a calibration curve of optic density versus dry biomass
lum of both organisms were acclimatized to carbon dioxide by (Reichert et al., 2006). Biomass carbon content was measured
maintaining them for 7 days under air mixed with 1% (v/v) added using a Perkin-Elmer 2400 CHNS (carbon, hydrogen, nitrogen
carbon dioxide. and sulfur) element analyzer calibrated to the 100% value using
a certified cystine standard (Perkin-Elmer, USA).
2.2. Photobioreactors and cultivation conditions
2.4. Kinetic parameters
Pure cultures of Spirulina sp. and S. obliquus were individ-
ually cultivated in three 2 L (1.8 L working volume) column Biomass (X) values and exponential regression were used to
photobioreactors (CPBR) connected in series (Fig. 1) and calculate the maximum specific growth rate (max , d1 ) dur-
labeled consecutively as CPBR1, CPBR2 and CPBR3. Agitation ing the logarithmic phase and maximum specific death rate (k,
and aeration were accomplished using air from a compressor d1 ) during the decline phase (Bailey and Ollis, 1986). The
doubling time (td , d) was calculated as td = ln 2(max )1 . The
maximum biomass concentration achieved in a photobioreac-
tor was designated Xmax (g L1 ). Productivity (P, g L1 d1 )
was obtained using the equation P = (Xt X0 )(t t0 )1 where
Xt is the biomass concentration (g L1 ) at t (d) and X0 is
the biomass concentration at inoculation (t0 ) (Schmidell et al.,
2001). Maximum productivity during cultivation was designated
Pmax (g L1 d1 ).

2.5. Carbon dioxide xation

Carbon dioxide fixation (F, g) by each organism was calcu-

lated from the CHNS biomass carbon content values.
The accumulation of fixed carbon dioxide (FA, g) in each
of the three individual photobioreactors which made up the
photobioreactor was calculated as FA = (Xt X0 )mcbm Vp (mCO2
mC 1 ), where FA can represent FA1, FA2 or FA3, where FA1,
Fig. 1. Three-stage serial photobioreactors scheme. All measurements in mil- FA2 and FA3 represent the accumulation of fixed carbon dioxide
limetres. in CPBR1, CPBR2 and CPBR3, respectively; Xt is the biomass
M.G. de Morais, J.A.V. Costa / Journal of Biotechnology 129 (2007) 439445 441

Table 1
Maximum specific growth rate (max , d1 ); maximum biomass concentration (Xmax , g L1 ); maximum productivity (Pmax , g L1 d1 ) for Spirulina sp. and S.
obliquus growing in the three different photobioreactors (CPBR) which constituted the three-stage serial photobioreactor, with and without carbon dioxide as the
sole carbon source
Carbon dioxide level (%, v/v)

Microalga and CPBR 0 6 12

max Xmax Pmax max Xmax Pmax max Xmax Pmax

Spirulina sp.
CPBR1 0.33 a 0.82 a 0.04 a 0.44 3.40 c 0.20 0.33 a 3.38 cf 0.17 e
CPBR2 0.33 a 0.85 a 0.14 b 0.27 abe 3.00 d 0.22 0.32 abe 3.20 0.17 e
CPBR3 0.29 ab 0.80 a 0.14 b 0.27 abe 2.90 d 0.19 0.29 ab 3.50 cf 0.18
S. obliquus
CPBR1 0.15 c 0.31 b 0.04 a 0.22 cf 1.56 0.10 d 0.22 cf 1.80 e 0.14 b
CPBR2 0.10 cd 0.27 b 0.06 ac 0.19 cfg 1.81 e 0.10 d 0.14 cdgh 1.50 g 0.10 d
CPBR3 0.07 d 0.24 b 0.05 a 0.19 cfg 1.36 0.07 c 0.14 cdgh 1.50 g 0.09 d

The air and, when present, carbon dioxide were fed serially into the photobioreactors starting with CPBR1. Values with the same letters in the same parameters
indicate that the values did not differ by the Tukey test at p 0.10.

concentration (g L1 ) at t (d) and X0 is the biomass concentra- in CPBR1, CPBR2 and CPBR3 at t + 1 (d); FA1t , FA2t and
tion at inoculation (t0 ); mcbm is the fraction of carbon in the FA3t represent the fixed carbon dioxide accumulated in CPBR1,
biomass (g g1 ); Vp (L) is the volume of culture medium in a CPBR2 and CPBR3 at time t = 1 d; m24 h represents the mass (g)
specific photobioreactor; mCO2 (g mol1 ) is the molar mass of of carbon dioxide added in 24 h.
carbon dioxide; mC (g mol1 ) the molar mass of carbon.
The total daily carbon dioxide fixation per gram of 2.6. Statistical analysis
carbon dioxide added to the medium (TF, g g1 ) was calcu-
lated for three-stage serial tubular photobioreactor as TF = The experimental results were evaluated by comparison of
(FA1(t+1) + FA2(t+1) + FA3(t+1) ) (FA1t + FA2t + FA3t ) m24 h 1 , the growth curves and analysis of variance (ANOVA) of the
where FA1(t+1) , FA2(t+1) and FA3(t+1) represent the fixed carbon kinetic parameters, significance being tested by the Tukey test
dioxide accumulated in CPBR1, CPBR2 and CPBR3 at t + 1 at p 0.10.
(d), respectively. FA1t , FA2t and FA3t represent the fixed
carbon dioxide accumulated in CPBR1, CPBR2 and CPBR3 at 3. Results
time t (d); m24 h represents the mass of carbon dioxide (g) added
in 24 h. The maximum total daily carbon dioxide fixation was The maximum specific growth rate and maximum biomass
designated MDF. concentration values for S. obliquus and Spirulina sp. growing
The mean carbon dioxide fixation in grams of total carbon in the three-stage serial tubular photobioreactors in the presence
dioxide fixed per gram of total carbon dioxide added to the of three different concentrations of carbon dioxide are shown in
media (MF, g g1 ) for three-stage serial tubular photobiore- Table 1 and the growth curves of biomass versus time are shown
actor was calculated as MF = (FA1(t+1) + FA2(t+1) + FA3(t+1) ) in Figs. 24.
(FA1t + FA2t + FA3t ) [m24 h (t + 1)]1 , where FA1(t+1) , FA2(t+1) In the absence of carbon dioxide Spirulina sp. runs biomass
and FA3(t+1) represent the fixed carbon dioxide accumulated increased until day 5 of cultivation (Fig. 2a), with no signif-

Fig. 2. Time course of biomass concentration for Spirulina sp. (a) and Scenedesmus obliquus (b) cultive in the absence of carbon dioxide in three-stage serial tubular
photobioreactor, where CPBR1 (), CPBR2 () and CPBR3 (+).
442 M.G. de Morais, J.A.V. Costa / Journal of Biotechnology 129 (2007) 439445

Fig. 3. Time course of biomass concentration for Spirulina sp. (a) and S. obliquus (b) using air supplemented with 6% (v/v) of carbon dioxide added to three-stage
serial tubular photobioreactor, where CPBR1 (), CPBR2 () and CPBR3 (+).

icant difference (p > 0.6004) between the maximum biomass as for Spirulina sp., but was only 0.06 g L1 d1 for CPBR2 and
concentration values for the three photobioreactors (Table 1). 0.05 g L1 d1 for CPBR3, lower than the corresponding values
Under these conditions the Spirulina sp. Xmax values (g L1 ) for Spirulina sp.
were significantly smaller (p < 0.0002) than those for the 6% In the Spirulina sp. runs with 6% carbon dioxide (Fig. 3a)
and 12% carbon dioxide runs (Table 1) but there was no sig- the Xmax values were high and there were significant dif-
nificant difference between the maximum productivity (Pmax , ferences (p 0.0002) between the different photobioreactors
g L1 d1 ) of Spirulina sp. in the different photobioreactors, (Table 1), with the highest value being obtained in CPBR1 which
with CPBR1 giving a Pmax value of 0.04 g L1 d1 and CPBR2 received the largest amount of carbon dioxide because it was
and CPBR3 both giving 0.14 g L1 d1 . The max values were the first in the series (Table 1). During the 21 days of culti-
similar for all three photobioreactors but after 5 days cultivation vation the concentration of Spirulina sp. biomass increased in
in the absence of carbon dioxide the Spirulina sp. biomass the three serial photobioreactors and the Pmax values for Spir-
values fell abruptly and the specifics death rates (k, d1 ) were ulina sp. in CPBR1, CPBR2 and CPBR3 were 0.20, 0.22 and
0.36, 0.22 and 0.43 d1 for CPBR1, CPBR2 and CPBR3, 0.19 g L1 d1 , respectively. The max values for Spirulina sp.
respectively, with the stationary phase being reached after cultivated in CPBR1 was significantly higher (p < 0.0002) than
10 days. that achieved in CPBR2 and CPBR3 which both showed the
At the same carbon dioxide level, the S. obliquus biomass same max values (Table 1).
concentration also increased until day 5 (Fig. 2b) but the Xmax The S. obliquus runs with 6% carbon dioxide (Fig. 3b) showed
values were significantly smaller (p < 0.0002) than those for significantly higher (p < 0.0002) Xmax values in CPBR2 than in
Spirulina sp. and S. obliquus max values were also signifi- CPBR1 and CPBR3 (Table 1). The S. obliquus Pmax values were
cantly lower than the corresponding values for Spirulina sp. 0.10 g L1 d1 in CPBR1 and 0.07 g L1 d1 in CPBR2 and
and there was a significant difference (p = 0.0015) between CPBR3, significantly lower (p < 0.0002) than the corresponding
CPBR1 and CPBR3 (Table 1). Under the same conditions, the values for Spirulina sp. The max value for S. obliquus was
S. obliquus Pmax value for CPBR1 was 0.04 g L1 d1 , the same highest in CPBR1, although there was no statistically significant

Fig. 4. Time course of biomass concentration for Spirulina sp. (a) and S. obliquus (b) using air supplemented with 12% (v/v) carbon dioxide added three-stage serial
tubular photobioreactor, where CPBR1 (), CPBR2 () and CPBR3 (+).
M.G. de Morais, J.A.V. Costa / Journal of Biotechnology 129 (2007) 439445 443

21 days) or 12% (initial pH 7.08 0.29 rising to 8.76 0.15

in 21 days) carbon dioxide runs. When S. obliquus was culti-
vated without carbon dioxide the initial pH was 6.95 0.03 but
increased to pH 9.18 0.26 after 24 h and reached a maximum
of about pH 10.03 0.64 after 15 days, similar to the 6% (initial
pH 5.40 0.01 rising to 8.62 0.39 in 21 days) and 12% (initial
pH 5.33 0.05 rising to 9.09 0.77 in 21 days) carbon dioxide
These results may have been due to the fact that in the absence
of carbon dioxide there was no formation of carbonic acid and
no abrupt decreases in pH. However, when carbon dioxide was
present the carbonic acid formed reduced the pH of the media
but this began to increase as the carbon dioxide was metabo-
lized by the microorganisms, which had been acclimatized to
1% carbon dioxide. Although no attempt was made to maintain
the organisms at the most appropriate pH for optimum growth
Fig. 5. Mean carbon dioxide fixation in three serial photobioreactors in the this did not appear to have any adverse effects on the growth,
presence of 6% (v/v) carbon dioxide to Spirulina sp. () and S. obliquus () indicating that both these organisms are resistant to variations
and with 12% (v/v) carbon dioxide to Spirulina sp. (), S. obliquus (). in pH.

difference between this value and the corresponding values for 4. Discussion
CPBR2 and CPBR3.
With 12% carbon dioxide the Spirulina sp. Xmax and max When fossil fuels are burned they produce about 12% of
values were significantly higher (p < 0.0002) than those for S. carbon dioxide (Lee et al., 2002) and it has been reported that
obliquus (Table 1, Fig. 4a). For S. obliquus cultivated using the cultivation of microalga could fix carbon dioxide equivalent to
same carbon dioxide concentration (Fig. 4b) Xmax was signifi- 500 MW of energy (Kadam, 2002). Our results show that both
cantly higher (p < 0.0002) in CPBR1 as compared with CPBR2 Spirulina sp. and S. obliquus can grow in media supplied with
and CPBR3, which both showed the same values (Table 1). air containing this concentration of carbon dioxide, indicating
The mean carbon dioxide biofixation rates (MF) in the three that these organisms could be used to sequester the carbon diox-
serial photobioreactors for Spirulina sp. and S. obliquus growing ide produced by combustion gases from thermoelectric power
on 6% and 12% carbon dioxide are shown in Fig. 5. In CPBR2 stations.
and CPBR3 the cyanobacterium Spirulina sp. presented signif- The maximum mean carbon dioxide biofixation rates values
icantly higher (p = 0.0002) MF values than did the microalgae for Spirulina sp. were recorded on day 7 with 6% carbon dioxide
S. obliquus, but for CPBR1 there was no significant difference and day 14 with 12% carbon dioxide, while for S. obliquus the
between the MF values for Spirulina sp. growing on 6% and maximum MF values occurred on day 8 with 6% carbon dioxide
12% carbon dioxide and S. obliquus growing on 6% carbon and day 9 with 12% carbon dioxide.
dioxide (data not shown). The highest Spirulina sp. MF values The growth rate of the diatom Chaetoceros wighamii (Araujo
(27.1437.9%) occurred in the 6% carbon dioxide runs, with and Garcia, 2005) and the microalga Tetraselmis (Olaizola et
the lowest mean carbon dioxide biofixation rates values in these al., 1991) increased when carbon dioxide was added to their
runs being higher than the highest values in all other runs. The growth medium, indicating that carbon dioxide can be a limiting
MF value for S. obliquus cultivated with 6% carbon dioxide was factor for the growth of a variety of different photosynthetic
7.4013.45%. In the 12% carbon dioxide runs the Spirulina sp. microorganisms. Chang and Yang (2003) cultivated Chlorella
MF value was 6.7017.06% while for S. obliquus it varied from strains NTU-H15 and NTU-H25 and found that the addition of
4.39% to 8.63%. Maximum daily carbon dioxide biofixation carbon dioxide stimulated the growth of the cells and resulted in
(MDF) for Spirulina sp. was 53.29% on day 7 and 45.61% on day high productivity of 0.31 g L1 d1 , with both strains showing
14 when growing on 6% and 12% carbon dioxide, respectively, the best results with 5% carbon dioxide.
while the corresponding values for S. obliquus were significantly Sung et al. (1999) grew Chlorella strain KR-1 and reported
lower (p 0.0541) at 28.08% on day 8 and 13.56% on day 9 different productivity with different levels of carbon dioxide
when growing on 6% and 12% carbon dioxide, respectively. In (10%, 1.1 g L1 d1 ; 30%, 0.8 g L1 d1 ; 50%, 0.6 g L1 d1
the absence of carbon dioxide biomass production ceased after and 70%, 0.1 g L1 d1 ), although these cultivations were not
5 days of cultivation. conducted in series. Hanaga et al. (1992) grew Scenedesmus and
The pH values in all three photobioreactors were very simi- Chlorella reported that both organisms presented a productivity
lar for both Spirulina sp. and S. obliquus at both carbon dioxide of 0.15 g L1 d1 with 10% carbon dioxide and 0.18 g L1 d1
concentrations. For Spirulina sp. the average pH of the three with 40% carbon dioxide. The concentration of carbon dioxide
serial photobioreactors was higher (9.70 0.20 to 11.71 0.50) in a culture media should never be less that required for the max-
in the absence of carbon dioxide than in the 6% (initial pH imum growth rate of the culture but should not be so high as to
7.16 0.16, then 9.33 0.05 after 24 h and 10.22 0.61 in exceed the maximum tolerated by a particular organism, thus
444 M.G. de Morais, J.A.V. Costa / Journal of Biotechnology 129 (2007) 439445

avoiding reduced fixation and/or loss to the atmosphere (Cheng the highest maximum productivity was 0.22 g L1 d1 , both in
et al., 2006). the presence of 6% carbon dioxide. Maximum daily carbon
Although the two organisms behaved differently regarding dioxide biofixation for Spirulina sp. was 53.29% and 45.61%
the carbon fixation, there was no obvious lag phase in any of when growing on 6% and 12% carbon dioxide, respectively,
the runs (Figs. 24), possibly because both the Spirulina sp. and while the corresponding values for S. obliquus were significantly
S. obliquus cultures used for the inoculum had been preadapted (p 0.0541) lower at 28.08% and 13.56% when growing on 6%
to carbon dioxide. Travieso et al. (2001) preadapted Spirulina and 12% carbon dioxide, respectively. In the absence of carbon
platensis cultures to carbon dioxide but still found a lag phase of dioxide biomass production ceased after 5 days of cultivation.
2 days. Yun et al. (1997) reported that cultures of the microalga The highest mean carbon dioxide fixation rates value was 37.9%
Chlorella vulgaris which had been pre-adapted to 5% carbon to Spirulina sp. in the 6% carbon dioxide runs.
dioxide grew better in the presence of 15% carbon dioxide as
compared to unadapted cultures and Lee et al. (2002) found a Acknowledgements
prolonged lag phase in unadapted Chlorella cultures when the
carbon dioxide concentration was increased from 10% to 70%. The authors thank the Centrais Eletricas Brasileiras S.A.
For Spirulina sp. and S. obliquus the highest average pH (ELETROBRAS) and the Companhia de Geracao Termica de
were 11.71 0.50 and 10.03 0.64, respectively, in the absence Energia Eletrica (CGTEE) for financially supporting this work.
of carbon dioxide, and the lower value pH were 7.08 0.29
and 5.33 0.05, respectively, in the 12% carbon dioxide runs.
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with carbon dioxide maintained constant growth at pH 4.2 but Araujo, S.C., Garcia, V.M.T., 2005. Growth biochemical composition of the
was completely inhibited at pH 3.5 (Sung et al., 1999) but diatom Chaetoceros cf. wighamii brightwell under different temperature,
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