Algal Research 12 (2015) 249–257

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Algal Research

journal homepage: www.elsevier.com/locate/algal

Operational strategies for maximizing CO2 utilization efficiency by the

novel microalga Scenedesmus obliquus SA1 cultivated in lab
scale photobioreactor
Samarpita Basu a,1, Abhijit Sarma Roy b,1, Aloke K. Ghoshal a,b,c, Kaustubha Mohanty a,b,c,⁎
a
Centre for Environment, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India
b
Centre for Energy, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India
c
Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India

a r t i c l e i n f o a b s t r a c t

Article history: The present study deals with optimization of operational parameters to maximize the CO2 utilization efficiency
Received 3 June 2015 by microalga: Scenedesmus obliquus SA1 (KC733762) previously isolated in our laboratory. SA1 strain was culti-
Received in revised form 7 September 2015 vated in a cylindrical glass photobioreactor under 15% CO2 concentration at various operational conditions. At a
Accepted 11 September 2015
light intensity of 4351 lx, a CO2 sparging duration of 12 h per day and flow rate of 0.43 L per hour, a maximum
Available online xxxx
biomass concentration of 3.32 ± 0.022 g L−1, a maximum specific growth rate of 1.24 ± 0.028 d−1, and a max-
Keywords:
imum CO2 utilization efficiency of 10.23% were obtained, which were higher than most of the relevant literature
Microalgae reports. Total inorganic carbon and total organic carbon concentrations of the cultivation medium were moni-
CO2 utilization efficiency tored with time, which showed increasing trends with an increase in biomass concentration.
Flue gas © 2015 Elsevier B.V. All rights reserved.
Open system

1. Introduction
The sharp rise in the level of atmospheric CO2 poses a great threat
of global warming and subsequent climate change. Conventional
coal-fired power stations release roughly 7% of total emitted CO 2
into the atmosphere. In general, the flue gases emitted from the
power plants consist of 10–20% CO2 [15]. There is thus an immediate
need to develop a sustainable and environment-friendly CO2 mitiga-
tion process. Photosynthetic organisms such as microalgae have
attracted much attention in recent times for this purpose since pho-
tosynthesis can be used to transport atmospheric carbon into a cycle
in which no additional CO2 is created [11]. Microalgae can grow
about 10–50 times faster than terrestrial plants thus achieving a
much higher CO2 fixation rate. Moreover, the microalgal biomass
produced through photosynthesis after CO 2 sequestration process
can be converted into a wide range of valuable products, such as
biofuels and food additives which represents additional benefits of
the microalgal CO2 mitigation process [9].
The microalgae culture system also plays an important role in the
CO2 sequestration process. Generally two types of microalgae culture
⁎ Corresponding author at: Centre for Environment, Indian Institute of Technology
Guwahati, Guwahati 781039, Assam, India.
E-mail address: kmohanty@iitg.ernet.in (K. Mohanty).
1
Both have contributed equally
systems are employed. These are open pond systems and closed
photobioreactor systems. In our previous study we have isolated
and identified a potential microalga Scenedesmus obliquus SA1
(KC733762) capable of growth and CO2 sequestration at 13.8 ±
1.5% CO2 and at elevated temperature of 40 °C [1]. SA1 proved to be
a potential candidate for CO2 sequestration from flue gas in closed
system. In the present study, we used the isolated SA1 for CO2 se-
questration in an open cylindrical glass photobioreactor under a
CO 2 concentration of 15% (typical flue gas concentration).The
photobioreactor was of open type so as to evaluate the growth kinet-
ic parameters and CO2 utilization efficiency of SA1 in an open culture
system. However, the photobioreactor used in this study was a lab-
scale experimentation system and did not correspond to the envi-
ronmental conditions and operational parameters associated with
an open pond system. The successful mitigation of CO2 using
microalgae requires that microalgae are sorted according to their
growth rates and CO2 utilization efficiencies. In this study, various
operational strategies were adopted to maximize the CO2 utilization
efficiency of the microalga SA1 at 15% CO2 concentration. Increased
height of the culture, intermittent CO 2 supply and reduced flow
rates were employed to prolong the residence time of CO 2 in the
open vessel. Since, in the open cultivation system, there is consider-
able diffusion of CO2 to the atmosphere, obtaining high CO2 utiliza-
tion efficiencies at 15% CO 2 concentration is quite challenging.
Literature reports suggest a decrease in CO2 utilization efficiencies
with an increase in inlet CO 2 concentration. Also, there have not

http://dx.doi.org/10.1016/j.algal.2015.09.010
2211-9264/© 2015 Elsevier B.V. All rights reserved.
250 S. Basu et al. / Algal Research 12 (2015) 249–257
been much reports of the CO2 fixation efficiencies at high CO2 con-
centrations in open cultivation system and mostly correspond to
closed photobioreactors.
2. Materials and methods
2.1. Organism and culture medium
S. obliquus SA1 (KC733762), a freshwater isolate of Guwahati, Assam
(26°11′15″N 91°45′4″E) was maintained in our laboratory in BG-11
medium (ATCC Medium 616) containing 1.5 g L− 1 sodium nitrate
(NaNO3), 0.04 g L− 1 dipotassium hydrogen phosphate (K2HPO4),
0.075 g L−1 magnesium sulfate (MgSO4·7H2O), 0.036 g L− 1 calcium
chloride (CaCl2·2H2O), 0.006 g L−1 citric acid (C6H8O7), 0.006 g L− 1
ferric ammonium citrate (C6H5 + 4yFexNyO7), 0.006 g L− 1 EDTA
(C10H14N2Na2O8·2H2O), 0.02 g L−1 sodium carbonate (Na2CO3), and
1 mL L−1 trace metal mix. The trace metal mix consisted of 2.86 g L−1
boric acid (H3BO3), 1.81 g L−1 manganese chloride (MnCl2·4H2O),
0.222 g L−1 zinc sulfate (ZnSO4·7H2O), 0.39 g L−1 sodium molybdate
(Na2MoO4·2H2O), 0.079 g L− 1 copper sulfate (CuSO4·5H2O) and
0.0494 g L−1 cobalt nitrate (Co(NO3)2·6H2O). The carbon free BG 11
media used in this study was devoid of Na2CO3 and had a pH of 6.9.
CO2 served as the sole carbon source for microalgal growth. All
inorganic chemicals were of analytical grade unless specified otherwise
and were obtained from Merck India; the CO2 and N2 cylinders (purity
99.99%) were procured from Assam Air Products Pvt. Ltd., Guwahati,
India.
2.2. Growth conditions
The photobioreactor (PBR) used for cultivation of the SA1 strain
consisted of an open cylindrical glass tube (0.05 m diameter, 0.6 m
length and 0.05 m diameter, 0.8 m length). Length to diameter ratio of
the working volume of the culture was varied (l/d ratio of 8, 10 and
12 was employed). Since highest biomass concentration was achieved
at l/d ratio 10 (0.05 m diameter, 0.5 m culture depth) (Fig. 1), all further
studies were carried out using a culture depth of 0.5 m in the PBR having
a diameter of 0.05 m (working volume of culture media being 860
mL).The operational parameters that were studied were varying light
intensities (2445, 2735, and 4351 lx), CO2 sparging duration (6, 12, 18
Fig. 1. Time course profile of biomass concentration for S. obliquus SA1 at varying l/d ratios.
and 24 h of CO2 supply), and CO2 flow rates (0.143, 0.43, 1.2 and
3.68 L per hour CO2). CO2 and N2 from gas cylinders were mixed and
the mixture gas was supplied via bubbling from the bottom of the PBR
to the culture at flow rates of 0.953, 2.86, 8 and 24.53 L per hour
(LPH) with CO2 flow rates of 0.143, 0.43, 1.2 and 3.68 LPH respectively
to obtain a final CO2 concentration of 15% in all the experiments. The
flow rates were maintained using pre-calibrated rotameters. For the
light intensity variation studies, continuous CO2 supply (24 h CO2) of
15% was provided at a flow rate of 1.2 LPH. For the intermittent CO2 sup-
ply studies, CO2 was supplied at a flow rate of 1.2 LPH and a light inten-
sity of 4351 lx was employed for the same. For the CO2 flow rate
variation studies, 18 h and 12 h intermittent CO2 sparging was done
and 4351 lx light intensity was employed. An inoculum size of
40 mg L−1 was used in all the experiments. The cultures were incubated
at 25 ± 1 °C and illuminated with daylight-type 20 W and 18 W fluores-
cent tubes. The light intensity on the surface of the PBR was measured
using a Digital Light Meter (model LX-101A, Lutron, Taiwan). 14 h of
light and 10 h of dark period was maintained during each experiment.
These growth conditions (culture temperature and photoperiod
regime) employed in our current study were optimized in our earlier
study [1].
2.3. Analysis of microalgal cell concentration
The cell concentration of the culture in the vessels was deter-
mined regularly by measuring optical density at a wavelength of
685 nm (OD685) using a UV/Vis spectrophotometer (model Evolution
201, Thermo Scientific, USA).The dry cell weight (DCW) of
microalgae biomass was obtained by filtering 5 mL aliquots of the
culture through previously weighed glass microfiber (GF/C, 37 mm
in diameter) filters (Whatman, England). Each loaded filter was
dried at 70 °C until the weight was invariant. The dry weight of the
blank filter was subtracted from that of the loaded filter to obtain
the microalgae dry cell weight. The OD685 values were converted to
biomass concentration via proper calibration between OD 685 and
dry cell weight and the conversion factor was determined (i.e., 1.0
OD685 approximately equals to 0.56 g DCW L− 1).
2.4. Determination of growth kinetic parameters and CO2 utilization
efficiency
Time-course profile of the biomass concentration (X; g L− 1) was
used to calculate the maximum specific growth rate (μmax, d−1). The
maximum biomass concentration achieved was designated as Xmax
(g L−1).
Overall biomass productivity (Poverall, mg L−1 d−1) was calculated
via the following equation:
ΔX
P overall ¼ ð1Þ
Δt
where ΔΧ is the variation of biomass concentration (mg L−1) within a
cultivation time of Δt (d).
Specific growth rate μ (d−1) was calculated from the following
equation:
ln ðX 1 =X 0 Þ
μ¼ ð2Þ
t 1 −t 0
where X1 and X0 were the biomass concentration (g L−1) on days t1 and
t0 respectively. The CO2 fixation rate (mg L− 1 d− 1) was determined
from the following equation:
 
−1
CO2 fixation rate ¼ 1:88  biomass productivity mg L−1 d : ð3Þ
 S. Basu et al. / Algal Research 12 (2015) 249–257 251

The CO2 utilization efficiency (%) was estimated from the following
equation [19]:
CO2 utilization efficiency ð%Þ  
−1
1:88  biomass productivity g L−1 d
¼    100:
−1
Input CO2 g CO2 L−1 d
The typical molecular formula of microalgal biomass, CO0.48H1.83
N0.11P0.01 [4] was used. Thus the factor 1.88 is the mass of CO2 fixed by
unit biomass, considering 51.39% of carbon in the dry biomass and
obtained as follows:
0:5139ð44=12Þ ¼ 1:88:
2.5. Determination of alkalinity and total organic carbon (TOC)
The cell suspension obtained from the light intensity variation stud-
ies was harvested daily and centrifuged at 6000 rpm for 10 min to pellet
down the microalgal cells. The supernatant obtained was thereafter
used for estimating the alkalinity and TOC. Alkalinity was determined
by the titration method as described by Sun et al. [16].
Briefly, the supernatant obtained was mixed with excess 0.1 N NaOH
solution. Subsequently, 10 mL of 20% BaCl2 solution was added. The so-
lution was shaken well and BaCO3 precipitate was formed. Then phenol-
phthalein indicator was added and the solution was titrated with 0.1 N
HCl till pink color disappeared. Next, methyl orange indicator was
added and again titration was done till pink color appeared. The amount
of HCl consumed was noted and the alkalinity was thereafter calculated.
TOC analysis of the supernatant was performed in a TOC analyzer
(model no. 1030, O-I-Analytical, Aurora, USA).
2.6. Statistical analysis
All the experiments were carried out in triplicate, and values were
expressed as mean ± standard deviation (SD). Statistical analysis was
carried out using Origin Pro 8.0.
3. Results and discussion
3.1. Effect of varying operational parameters on growth kinetic parameters
of SA1
The operational parameters were varied to investigate the most
ð4Þ suitable conditions for efficient CO2 sequestration by microalga SA1
from flue gas (containing CO2 in the range of 10–20%). Varying
light intensities (2445, 2735 and 4351 lx) were employed and 15%
CO2 was continuously fed into the microalgal culture at a flow rate
of 1.2 L per hour (LPH). Light intensity controls photosynthetic
growth in any microalgal system. It also affects CO2 removal rates,
biomass concentration and overall growth rate [15]. In the present
study it was found that SA1 showed improvement in all the growth
kinetic parameters with an increase in light intensity (Table 1). The
biomass concentration as a function of incubation time at varying
ð5Þ light intensities were plotted (Fig. 2a). The maximum biomass con-
centration (Xmax) (3.15 ± 0.065 g L− 1) obtained at a light intensity
of 4351 lx was 38% higher than Xmax obtained at 2445 lx (1.93 ±
0.009 g L− 1) and 17% higher than Xmax obtained at 2735 lx (2.61 ±
0.015 g L− 1). As evident from the literature reports [7,8], there is a
direct relationship between light intensity, growth rate, cell density
and biomass productivity. Furthermore, photosynthetic rate is di-
rectly proportional to light intensity below the light saturation
point, resulting in an increase in biomass productivity and CO2 up-
take. Also, increased light intensity leads to an increase in the ratio of
the photosynthetic pigments such as Chlorophyll a/b as well as caroten-
oid/chlorophyll total as an adaptation mechanism. George et al. [7] and
Cheirsilp and Torpee [3] have observed that increase in irradiance signif-
icantly enhanced the chlorophyll content in microalgae Ankistrodesmus
falcatus and Chlorella sp. respectively. This might be the case in S. obliquus
SA1 strain leading to increased photosynthetic efficiency at 2735 lx than
at 2445 lx resulting in 26% increase in biomass concentration and 20% in-
crease in CO2 fixation rate with a concomitant change in a light intensity
of 290 lx. Moreover, close light intensity values were chosen to study the
effects of small changes in light intensity on the growth kinetic parame-
ters of the reported microalga with minimal change in the total power
consumption.
Overall biomass productivity (Poverall), maximum specific growth
rate (μmax), maximum biomass productivity (Pmax), maximum CO2 fixa-
tion rate and maximum CO2 utilization efficiency were highest at a light
intensity of 4351 lx with values of 202.67 ± 1.31 mg L−1 d−1, 0.89 ±
0.004 d−1, 772.24 ± 7.77 mg L−1 d−1, 1451.81 ± 14.62 mg L−1 d−1

Table 1
Variation of growth kinetic parameters of Scenedesmus obliquus SA1 (data represents mean ± standard deviation).

Parameters studied Incubation Input Maximum Overall biomass Maximum specific Maximum biomass Maximum CO2 Maximum CO2
time CO2/day biomass productivity growth rate productivity fixation rate utilization
(days) (L) (Xmax) (Poverall) (μmax) (Pmax) (mg L−1 d−1) efficiency
(g L−1) (mg L−1 d−1) (d−1) (mg L−1 d−1) (%)

Light intensity (lx) 2445 15 28.8 1.93 ± 0.009 119.34 ± 0.254 0.78 ± 0.023 291.94 ± 35.33 548.85 ± 66.43 0.97
24 h CO2 supply 2735 15 28.8 2.61 ± 0.015 150.66 ± 0.814 0.88 ± 0.038 443.14 ± 5.93 833.11 ± 11.15 1.47
at 1.2 LPH 4351 15 28.8 3.15 ± 0.065 202.67 ± 1.31 0.89 ± 0.004 772.24 ± 7.77 1451.81 ± 14.62 2.57
CO2 sparging 6 8 7.2 0.50 ± 0.005 52.64 ± 0.546 1.11 ± 0.028 177.52 ± 6.31 333.73 ± 11.86 2.36
duration (h) 12 8 14.4 1.87 ± 0.006 193.76 ± 14.16 1.20 ± 0.031 558.88 ± 3.87 1053.50 ± 10.59 3.73
CO2 supply at 1.2 18 8 21.6 2.36 ± 0.029 222.20 ± 5.77 1.25 ± 0.034 592.29 ± 26.01 1113.51 ± 48.91 2.63
LPH 24 15 28.8 3.15 ± 0.065 202.67 ± 1.31 0.89 ± 0.004 772.24 ± 7.77 1451.81 ± 14.62 2.57
4351 lx
CO2 flow rate 0.43 8 7.74 1.85 ± 0.019 214.83 ± 2.68 0.97 ± 0.043 597.70 ± 52.73 1123.68 ± 99.14 7.40
(LPH) 1.2 8 21.6 2.36 ± 0.029 222.20 ± 5.77 1.25 ± 0.034 592.29 ± 26.01 1113.51 ± 48.91 2.63
18 h CO2 supply 3.68 14 66.24 4.29 ± 0.022 294.14 ± 1.24 1.07 ± 0.002 672 ± 5.60 1263.36 ± 10.52 0.97
4351 lx
CO2 flow rate 0.143 10 1.71 0.56 ± 0.002 53.51 ± 0.252 0.78 ± 0.018 125.81 ± 3.18 236.52 ± 5.98 7.03
(LPH) 0.43 13 5.16 3.32 ± 0.022 253.27 ± 1.25 1.24 ± 0.028 550.66 ± 28.18 1035.25 ± 52.98 10.23
12 h CO2 supply 1.2 8 14.4 1.87 ± 0.006 193.76 ± 14.16 1.20 ± 0.031 558.88 ± 3.87 1053.50 ± 10.59 3.73
4351 lx
252 S. Basu et al. / Algal Research 12 (2015) 249–257
Fig. 2. Time course profile of biomass concentration for S. obliquus SA1 at varying (a) light intensities, (b) CO2 sparging durations, (c) flow rates at 18 h per day CO2 supply and (d) flow rates
at 12 h per day CO2 supply.
and 2.57% respectively. Our result corresponded with that of Hulatt
and Thomas [10] who reported an increase in biomass concentra-
tion (3.62 g L − 1 N 3.18 g L − 1 N 2.7 g L − 1 ) and CO 2 fixation rate
(930 mg L− 1 d− 1 N 830 mg L− 1 d− 1 N 720 mg L− 1 d− 1) in microalga
Chlorella vulgaris with an increase in light intensity (50 W m − 3 N
20 W m− 3 N 10 W m− 3) under 4% CO2 supply. Thus, a light intensity
of 4351 lx was employed in all subsequent experiments.
Continuous gas sparging into the cultivation media in the open cul-
tivation system leads to considerable diffusion and escape of CO2 to the
atmosphere and thus less amount of CO2 is utilized by microalgae. To
further increase the CO2 utilization efficiency by microalga SA1, CO2
was supplied at varying time intervals: 6 h, 12 h and 18 h per day
(Table 1). A constant flow rate of 1.2 LPH was maintained. Time course
profile of biomass concentration at varying gas sparging times is shown
in Fig. 2b. The highest value of maximum biomass concentration (Xmax)
(3.15 ± 0.065 g L−1) was obtained at continuous gas supply of 24 h on
day 14 of cultivation (the results discussed in 4351 lux light intensity
studies). The highest value of maximum specific growth rate (μmax) of
1.25 ± 0.034 d− 1 was obtained at CO2 supply of 18 h per day. Tang
et al. [17] reported μmax value of 0.887 ± 0.012 d− 1 and 0.780 ±
0.089 d−1 in S. obliquus SJTU-3 supplied with 10% CO2 and 20% CO2 re-
spectively. The μmax value (1.25 ± 0.034 d−1) obtained at 15% CO2
supply (18 h per day) with S. obliquus SA1 was 1.4 and 1.6 times higher
respectively than the reports of Tang et al. [17].Also the maximum CO2
fixation rate (1113.51 ± 48.91 mg L−1 d−1) obtained at 15% CO2 supply
(18 h per day) with the SA1 strain was 3.8 and 4.5 times higher
respectively than that obtained by Tang et al. [17] at 10% (288 ±
0.004 mg L−1 d−1) and 20% (246 ± 0.002 mg L−1 d−1) CO2 concentra-
tion. The maximum biomass concentration (Xmax) obtained at
18 h per day CO2 supply (2.36 ± 0.029 g L−1) and 12 h per day CO2 sup-
ply (1.87 ± 0.006 g L−1) was 4.7 and 3.7 times higher respectively than
that obtained at 6 h per day CO2 supply (0.50 ± 0.005 g L− 1). SA1
showed increase in all the growth kinetic parameters with an increase
in the duration of CO2 sparging. 6 h per day of CO2 supply proved insuf-
ficient for biomass production and a maximum biomass productivity
(Pmax) of 177.52 ± 6.31 mg L− 1 d−1 was obtained which was 3.14
and 3.33 times lower than that obtained at 12 h/day (558.88 ±
3.87 mg L−1 d−1) and 18 h/day (592.29 ± 26.01 mg L−1 d−1) CO2 sup-
ply respectively. The maximum CO2 utilization efficiency (3.73%) by
SA1 was highest at 12 h per day of CO2 supply at 1.2 LPH. The maxi-
mum biomass productivity (P max) of 558.88 ± 3.87 mg L − 1 d − 1
obtained at 12 h per day of CO 2 supply was 7.35 and 3.60 times
higher than that reported by de Morais and Costa [5] (76 ±
0.001 mg L− 1 d− 1) and Tang et al. [17] (155 ± 0.004 mg L− 1 d− 1)
for S. obliquus strains cultivated under 12% and 10% CO2 supply re-
spectively. High biomass productivity could benefit downstream ap-
plications such bio-energy production in addition to CO2 biofixation.
Moreover, by decreasing the CO 2 sparging duration from 24 h to
 S. Basu et al. / Algal Research 12 (2015) 249–257 253

12 h per day the maximum CO2 utilization efficiency by SA1 could be The effect of varying flow rates of CO2 was studied at 18 h per day
increased from 2.57% to 3.73% indicating less CO 2 escape at and 12 h per day of gas supply to obtain the optimum flow rate in
12 h per day CO2 supply. which the retention time of CO 2 is increased leading to high

Fig. 3. Variation of CO2 fixation rate and CO2 utilization efficiency with incubation time at (a) 2445 lx, (b) 2735 lx, (c) 4351 lx, (d) 6 h per day CO2 supply, (e) 12 h per day CO2 supply,
(f) 18 h per day CO2 supply, (g) 0.43 LPH at 18 h per day CO2 supply, (h) 3.68 LPH at 18 h per day CO2 supply, (i) 0.43 LPH at 12 h per day CO2 supply and (j) 0.143 LPH at
12 h per day CO2.
254 S. Basu et al. / Algal Research 12 (2015) 249–257
Fig. 3 (continued).
maximum CO 2 utilization efficiency. Although the maximum bio-
mass concentration (Xmax ) was highest (4.29 ± 0.022 g L− 1 )
(Fig. 2c) at a CO2 flow rate of 3.68 LPH, the maximum CO2 utilization
efficiency (0.97%) was 7.6 times lower than that obtained at a flow
rate of 0.43 LPH CO2 (7.40%) at 18 h per day of gas supply. The max-
imum specific growth (μmax ) and maximum biomass productivity
(Pmax) obtained at 0.43 LPH CO2 supply were 0.97 ± 0.043 d− 1 and
597.70 ± 52.73 mg L− 1 which indicated fast growth, good biomass
productivity and efficient CO 2 utilization by the SA1 strain at low
flow rate of CO2. Flow rate variations of CO2 on growth kinetics and
CO2 utilization efficiency of SA1 were further studied at
12 h per day CO2 supply. At 0.43 LPH CO 2 supply continuously for
12 h per day, a maximum biomass concentration (Xmax) of 3.32 ±
0.022 g L − 1 was obtained after 13 days of cultivation (Fig. 2d).
Tang et al. [17] obtained the highest Xmax value of 1.84 ±
0.01 g L− 1 in S. obliquus SJTU-3 at a CO2 concentration of 10% after
a cultivation period of 14 days. This value was 1.8 times lower than
that obtained with SA1 after 13 day cultivation period. The maxi-
mum CO2 utilization efficiency value (10.23%) was also highest
under this condition. On decreasing the flow rate further to
0.143 LPH CO2 (12 h per day supply) the maximum biomass produc-
tivity (Pmax)and maximum biomass concentration (Xmax) decreased
to 125.81 ± 3.18 mg L − 1 d− 1 and 0.56 g L− 1 respectively. These
values were 77% and 83% lower respectively as compared to the
Pmax (550.66 ± 28.18 mg L − 1 d− 1) and Xmax (3.32 ± 0.022 g L− 1)
values obtained at 0.43 LPH CO 2 supply of 12 h per day (Table 1).
The maximum CO 2 utilization efficiency at 0.143 LPH
CO 2 (12 h per day supply) was 7.03% which was 1.45 times lower
than the highest value of maximum CO 2 utilization efficiency
(10.23%) obtained at 0.43 LPH CO2 (12 h/day supply). Also the max-
imum specific growth (μ max) of 1.24 ± 0.028 d− 1 obtained at
0.43 LPH CO2 (12 h per day supply) was 1.27 times higher than
μmax (0.97 ± 0.043 d − 1) value obtained at 0.43 LPH CO 2
(18 h per day supply). Thus, the optimum condition for obtaining
high biomass productivity and fast growth rate of SA1 along with
high maximum CO2 utilization efficiency was inferred as: 15% CO2
supply at 0.43 LPH for 12 h per day at a light intensity of 4351 lx
with light regime of 14 h:10 h (light:dark). SA1 with its high biomass
yield, fast growth rates and improved CO2 utilization efficiency (in
open cultivation system) at high CO2 concentration thus proved to
be a potential strain for CO2 biofixation from industrial flue gas.
3.2. Periodic variations of CO2 fixation rate and CO2 utilization efficiency in
SA1
The variation of the daily CO2 fixation rate and CO2 utilization effi-
ciency under various operational conditions at 15% inlet CO2 concentra-
tion were plotted (Fig. 3a–j). The maximum CO2 fixation rate
 S. Basu et al. / Algal Research 12 (2015) 249–257 255

(mg L−1 d−1) and maximum CO2 utilization efficiency (%) were highest (236.52 ± 5.98 mg L−1 d−1) (Fig. 3j) which were 5.92 and 4.38 times
at a light intensity of 4351 lx (24 h continuous CO2 supply at 1.2 LPH) lower respectively than that obtained at 0.43 LPH CO2 supply of
and the values were 1451.81 ± 14.62 mg L−1 d−1 and 2.57% respective- 12 h per day (Table 1). However the maximum CO2 utilization efficiency
ly obtained on day 5 of incubation (Fig. 3c).The maximum CO2 fixation of 7.03% obtained under this condition was only 1.46 times lower than
rate (1451.81 ± 14.62 mg L−1 d−1) obtained was 62% higher than that that obtained at 0.43 LPH CO2 supply of 12 h per day. Thus CO2 supply
obtained at a light intensity of 2445 lx (548.85 ± 66.43 mg L−1 d−1)and at a very low flow rate of 0.143 LPH proved insufficient for microalgal
42% higher than that obtained at a light intensity of 2735 lx (833.11 ± growth and biomass production. Decreasing the flow rate of CO2 from
11.15 mg L−1 d−1). Also the maximum CO2 fixation rate (2.57%) was 1.2 LPH (which was also employed in our earlier studies: [1,2]) to
2.65 times higher than that obtained at a light intensity of 2445 lx 0.43 LPH (inlet CO2 concentration being maintained constant at 15%)
(0.97%) and 1.75 times higher than that obtained at a light intensity of and reducing the CO2 sparging duration to 12 hCO2 per day thus proved
2735 lx (1.47%). On decreasing the CO2 sparging duration (at 1.2 LPH) to be the ideal condition for CO2 sequestration by S. obliquus SA1. In
from 24 h to 12 h per day, the maximum CO2 utilization efficiency ob- order to further improve CO2 utilization efficiency, the relationship be-
tained on day 4 (3.73%) was 1.45 times higher (2.57% for 24 h supply tween the time with and without sparging, CO2 dissolution rate, and
at 1.2 LPH) (Fig. 3e). The maximum CO2 fixation rate under 12 h and CO2 utilization rate requires further study outside of the bounds of this
18 h of CO2 supply at a flow rate of 1.2 LPH was obtained on day 4 of in- work. During the stopping time (i.e, when there is no gas sparged), the re-
cubation and the values were 1053.50 ± 10.59 mg L−1 d−1 and maining CO2 (including CO2 in culture and residual gas) should be ade-
1113.51 ± 48.91 mg L− 1 d− 1 respectively (Fig. 3e and f). Although quate for microalgal growth, so that the residual CO2 and DIC in the
the maximum CO2 fixation rate obtained at 6 h per day of CO2 supply medium could be completely utilized during the remaining hours of
(333.73 ± 11.86 mg L− 1 d−1) (Fig. 3d) was 3.15 times lower than light. These parameters are vital for designing a system for maximal CO2
that obtained at 12 h per day CO2 (1053.50 ± 10.59 mg L−1 d−1), the utilization from the input stream as well as for maximum microalgal
maximum CO2 utilization efficiency under the same condition (2.36%)
was only 1.58 times lower than that obtained at 12 h per day CO2 supply
(3.73%).
The flow rate of CO2 was decreased to 0.43 LPH at 18 h per day of CO2
supply which increased the retention time of CO2 in the culture increas-
ing the maximum CO2 utilization efficiency to 7.40% on day 6 (Fig. 3g)
which was 2.81 times higher than that (2.63%) obtained at 1.2 LPH.
The maximum CO2 fixation rate (1123.68 ± 99.14 mg L−1 d−1) obtain-
ed under the same condition was also higher than that (1113.51 ±
48.91 mg L−1 d−1) obtained at 1.2 LPH (18 h per day supply). On in-
creasing the flow rate of CO2 to 3.68 LPH, although the maximum CO2
fixation rate (1263.36 ± 10.52 mg L− 1 d− 1) obtained on day 5
(Fig. 3h) was high due to the high biomass concentration achieved at
high CO2 flow rate, much CO2 escaped from the system and the value
of maximum CO2 utilization efficiency (0.97%) was 7.62 times lower
than that obtained at a low flow rate of 0.43 LPH (7.40%)
(Table 1).Thus supplying more CO2 to the system (66.24 L per day)
(Table 1) keeping the concentration constant at 15% does not prove to
be economical since much of the gas remains unutilized by microalgae.
The flow rate of 0.43 LPH was employed at CO2 supply of 12 h per day to
study the utilization efficiency of strain SA1 under this condition. Max-
imum CO2 utilization efficiency increased to 10.23% at 0.43 LPH CO2
supply for 12 h per day (Fig. 3i). Also high maximum CO2 fixation rate
(1035.25 ± 52.98 mg L−1 d−1) and maximum biomass concentration
(3.32 ± 0.022 g L−1) were obtained under this condition. A maximum
CO2 utilization efficiency value of 10.23% in the open cultivation system
under 15% inlet CO2 concentration was 3.96 times higher than the value
(2.58%) reported by De Morais and Costa [6] who cultivated S. obliquus
in vertical tubular photobioreactor (closed system) under 12% inlet con-
centration. Westerhoff et al. [18] reported maximum CO2 utilization
efficiency of 3% by Scenedesmus + mixed culture under inlet CO2 con-
centration of 12% in a helical tubular photobioreactor. This value was
3.41 times lower than that achieved by SA1 in the open system. Lv et al.
[13] reported a decrease in the maximum CO2 biofixation ratio (%) from
35% to 2% with an increase in inlet CO2 concentration from 0.5% to 12%
in microalga C. vulgaris cultivated cylindrical photobioreactor. The maxi-
mum CO2 utilization efficiency (10.23%) obtained with SA1 under 15%
inlet CO2 concentration supplied at a flow rate of 0.43 LPH for
12 h per day was 5 times higher than the value reported by Lv et al. at
12% inlet CO2 concentration. Thus, SA1 proves to be a promising strain
for CO2 sequestration in open cultivation system since it shows high
CO2 utilization efficiency (10.23%) as compared to literature reports at
15% inlet CO2 concentration. When the flow rate of CO2 was decreased
further to 0.143 LPH (1.71 L CO2 per day) with 12 h per day supply,
there was a drastic drop in the maximum biomass concentration Fig. 4. Variation of (a) alkalinity and (b) total organic carbon (TOC) concentration in cul-
(0.56 ± 0.002 g L−1) (Fig. 2d) and maximum CO2 fixation rate tivation medium at varying light intensities with incubation time.
256 S. Basu et al. / Algal Research 12 (2015) 249–257
biomass production and our ongoing studies are focused on optimizing
these parameters and will be reported in our subsequent communication.
3.3. Analysis of alkalinity and TOC in cultivation media
Alkalinity (mol L−1) and TOC (ppm) values of the culture superna-
tant were obtained for the light intensity variation studies to investigate
the variations of these parameters with continuous CO2 supply of 15%.
Alkalinity values in the culture medium increased with progress in incu-
bation time in all the 3 different light intensity studies until day 12 of in-
cubation (Fig. 4a). These values were 0.0175 mol L−1, 0.02 mol L−1 and
0.0225 mol L−1 respectively for a light intensity of 2445, 2735 and
4351 lx. Alkalinity values increased in the culture due to consumption
of nitrate by microalgae, which in turn released hydroxyl ions that
remained in the medium leading to increased alkalinity. After day 12,
CO2 utilization might have decreased as nitrate was no longer available
for consumption. This should have caused acidification of the culture,
and thus decreased values of alkalinity were obtained since the alkalin-
ity test is pH based.
The amount of CO2 taken up by SA1 on day 14 of incubation was
0.082 mol L− 1, 0.111 mol L− 1, and 0.134 mol L− 1 respectively at a
light intensity of 2445, 2735 and 4351 lx. (Fig. 5a–c). The biomass con-
centration on the 12th day of incubation was 1.47 ± 0.016 g L−1, 1.85 ±
0.007 g L−1 and 2.75 ± 0.016 g L−1 for light intensities of 2445, 2735
and 4351 lx respectively (Fig. 2a). Peltier and Thibault [14]
Fig. 5. Variation of alkalinity in cultivation medium and CO2 uptake by SA1 at (a) 2445 lx, (b) 2735 lx and (c) 4351 lx.
demonstrated that respiration (especially photorespiration) of
microalgae partly promotes the dissolved inorganic carbon (DIC) or
CO2 concentration in water. Increased biomass concentration of
microalgae at a light intensity of 4351 lx might have led to increased
respiration leading to increased alkalinity value in the cultivation
media. Also, the biomass concentration obtained at 2445 lx was lower
than that obtained at 2735 lx leading to increased alkalinity levels in
the cultivation media at 2735 lx. The TOC values in the cultivation
media also showed increasing trends with an increase in light intensity
values. The highest value of TOC was obtained on day 15 of cultivation in
all the cases. These were 182.32 ppm (4351 lx) N 69.34 ppm
(2735 lx) N 46.69 ppm (2445 lx) (Fig. 4b). The biomass concentration
on the same day was 3.08 ± 0.020 g L−1 (4351 lx) N 2.30 ±
0.014 g L−1 (2735 lx) N 1.83 ± 0.004 g L−1 (2445 lx) (Fig. 2a). Generally
microalgae can release some photosynthetically fixed organic carbon
into the surrounding water through the leaky algal cells which resulted
from physical stress [12]. Since high microalgal biomass was generated
at a light intensity of 4351 lx, photosynthetically fixed organic carbon
was also highest at 4351 lx which must have been released in the cul-
ture media with an increase in incubation time resulting in increased
TOC concentration in the media at 4351 lx. The initial pH of the BG-11
medium employed for microalgal cultivation was 6.94. A detailed
study of the variation in medium pH with an increase in microalgal bio-
mass was reported in our earlier publication [1]. The initial pH of the
medium was 6.94 which gradually became alkaline (reaching about
 S. Basu et al. / Algal Research 12 (2015) 249–257
8.49 on day 34 of cultivation) with an increase in microalgal biomass
concentration. It is an established fact that microalgae can uptake both
CO2 and HCO− −
3 through the cell membrane leaving OH in the culture
medium which in turn increases the pH of the medium [20]. This fact
was evident from our earlier experimental finding.
4. Conclusions
Microalga strain S. obliquus SA1 was cultivated in lab scale
photobioreactor and operational parameters were varied to obtain the
optimum condition for increased CO2 utilization and biomass produc-
tion. 15% CO2 supply at 0.43 LPH for 12 h per day proved to be the
ideal condition at which a maximum CO2 utilization efficiency of
10.23% was achieved which was higher than most of the relevant stud-
ies. Also high maximum biomass concentration (3.32 ± 0.022 g L−1)
and maximum CO2 fixation rate (1035.25 ± 52.98 mg L−1 d−1) were
obtained under this condition making SA1 a promising strain for CO2
mitigation from flue gas.
Acknowledgment
This work was financially supported by the Council of Scientific and
Industrial Research (CSIR) (24(0320)/12/EMR-II, dated: 07-05-2012),
New Delhi, Government of India, India.
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