Ecological Engineering 58 (2013) 142–148

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Cultivation of microalgae species in tertiary municipal wastewater

supplemented with CO2 for nutrient removal and biomass production
Min-Kyu Ji a , Reda A.I. Abou-Shanab a,b , Seong-Heon Kim a,∗∗ , El-Sayed Salama a ,
Sang-Hun Lee c , Akhil N. Kabra d , Youn-Suk Lee e , Sungwoo Hong f , Byong-Hun Jeon a,∗
a
Department of Environmental Engineering, Yonsei University, Wonju 220-710, South Korea
b
Department of Environmental Biotechnology, City of Scientific Research and Technology Applications, New Borg El Arab City, Alexandria 21934, Egypt
c
Research Division for Industry and Environment, Korea Atomic Energy Research Institute, Jeongeup, Jeollabuk-do 580-185, South Korea
d
Department of Biotechnology, Shivaji University, Kolhapur 416 004, India
e
Department of Packaging, Yonsei University, Wonju 220-710, South Korea
f
National Forensic Service, Seoul 158-097, South Korea

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

Article history: This study demonstrates the potential for algae-based biofuel production by coupling advanced wastewa-
Received 23 January 2013 ter treatment with microalgae cultivation for low-cost lipid production. Three species (Chlorella vulgaris,
Received in revised form 30 April 2013 Scenedesmus obliquus and Ourococcus multisporus) with higher biomass yield were selected and cultured
Accepted 8 June 2013
in wastewater amended with 15% CO2 . C. vulgaris, S. obliquus and O. multisporus showed optimal specific
growth rates (opt ) of 1.37, 1.14 and 1.00 day−1 , respectively, and almost complete removal (>99%) of
Keywords:
nitrogen and phosphorus within 4 days. The highest specific lipid productivity was 0.164 g-lipids g-cell−1
Biodiesel
day−1 and oleic acid was increased to 44% in C. vulgaris after 7 days of cultivation in the presence of CO2 .
Chlorella vulgaris
Domestic wastewater
It was concluded that C. vulgaris is a good potential source for the production of biodiesel coupled with
CO2 nutrient removal from wastewater.
Biomass yield © 2013 Elsevier B.V. All rights reserved.
Nutrient uptake

1. Introduction
The world has been confronted with an energy and water
crisis associated with the depletion of fossil fuels and freshwa-
ter, coupled with an atmospheric accumulation of greenhouse
gases that cause global warming (Rodolfi et al., 2009). Renew-
able sources of energy and water are required to address these
depletions. Microalgae have attracted a great deal of attention as
biofuel feedstock due to their high oil yield (5,000–100,000 L/ha-y)
and their ability to capture waste CO2 and to be cultivated in
brackish, salt or wastewaters (Levine et al., 2010). A variety of
microalgae species (Ourococcus multisporus, Nitzschia cf. pusilla,
Chlamydomonas mexicana, Scenedesmus obliquus, Chlorella vulgaris
and Micractinium reisseri) have been cultured in various water
sources (i.e., fresh and wastewater) and different CO2 conditions
(Ho et al., 2010; Abou-Shanab et al., 2013). Carbon dioxide is an
important factor for microalgal growth and the biosynthesis of
∗ Corresponding author. Tel.: +82 33 760 2446; fax: +82 33 760 2571.
∗∗ Corresponding author. Tel.: +82 33 760 2380; fax: +82 33 760 2571.
E-mail addresses: seongheo@yonsei.ac.kr (S.-H. Kim), bhjeon@yonsei.ac.kr
(B.-H. Jeon).
fatty acids. Approximately 1.8 kg of CO2 is required for the pro-
duction of 1 kg of algal biomass (Chisti, 2007). Flue gases generated
from power plants containing 10–15% (v/v) CO2 can be used for
microalgal cultivation (Zeiler et al., 1995). Successful cultivation
of Scenedesmus sp., Chlorella sp. and Nannochloropsis sp. has been
reported the relatively high microalgal growth rates using 10–15%
flue or synthetic CO2 (Lee et al., 2002; Jin et al., 2006; Jiang et al.,
2011).
Although the nutrient-rich wastewater growth media used in
previous studies have shown great potential for microalgal growth,
nutrient-rich raw wastewaters produced from animal-related
industries (e.g., livestock wastewater) have several disadvantages,
including high concentrations of ammonium ion, turbidity, chro-
maticity and unbalanced C:N:P ratios, for which a large amount
of diluent is required for pretreatment (Wang et al., 2010b; Ji
et al., 2012). Several studies have reported the application of sec-
ondary treated domestic wastewater containing relatively low
levels of inorganic constituents. These studies have mainly focused
on improving nutrient removal and decreasing the nutrient loads
deposited in water bodies along with simultaneous lipid accu-
mulation in microalgae (Wang et al., 2010a; Cho et al., 2011).
Microalgae-based treatment processes using a wide variety of
wastewaters have been investigated for simultaneous nutrient

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http://dx.doi.org/10.1016/j.ecoleng.2013.06.020
 M.-K. Ji et al. / Ecological Engineering 58 (2013) 142–148 143

Table 1 Three microalgal species (O. multisporus, C. vulgaris and S. obliquus)

Physicochemical characteristics of tertiary-treated municipal wastewater.
were finally selected for further study after preliminary analyses of
Parameter Concentration their biomass yield.
−1
Total carbon (mg L ) 22.6 ± 1
Total inorganic carbon (mg L−1 ) 14.6 ± 0.1 (51.7 ± 0.4)a
Total organic carbon (mg L−1 ) 8.1 ± 0.2
2.2. Wastewater sampling and analysis
pH 7.3 (5.7)a
TN (mg L−1 ) 8.7 ± 0.5 Tertiary-treated municipal wastewater was collected from
NH4 -N (mg L−1 ) 0.4 ± 0.1 municipal wastewater treatment plant at Wonju, South Korea. The
NO3 -N (mg L−1 ) 8.5 ± 0.4
wastewater effluent sample was immediately filtered using 0.2 ␮m
NO2 -N (mg L−1 ) NDb
TP (mg L−1 ) 1.71 ± 0.3 nylon microfilters to remove microorganisms and fine suspended
PO4 -P (mg L−1 ) 1.69 ± 0.4 particles. The standard methods 4500-N C, 4500-NH3 G and 4500
N/P molar ratio 11:1 P. B. 5 were used to determine the T-N, NH4 -N and T-P in water
Alkalinity (mg as CaCO3 L−1 ) 50.2 ± 1.8 samples, respectively (APHA, 1998). The concentrations of bromide
Conductivity (␮S cm−1 ) 590
ORP (mV) 215.5
(Br− ), chloride (Cl− ), nitrite (NO2 − ), nitrate (NO3 − ), phosphate
Suspended solid (mg L−1 ) 0.02 (PO4 3− ) and sulfate (SO4 2− ) were determined by single-column
Cl− (mg L−1 ) 75 ± 0.4 ion chromatography (Metrohm 850 professional IC, Switzerland).
SO4 2− (mg L−1 ) 8.0 ± 0.5 Metal ions were analyzed using an ELAN DRC II inductively cou-
Metallic ions (mg L−1 )
pled plasma-mass spectrophotometer (ICP-MS, PerkinElmer SCIEX,
Ca2+ 31.4 ± 0.5
Na+ 22.7 ± 0.9 USA) after acidification with 1% (v/v) nitric acid. The suspended
K+ 10.8 ± 0.2 solid concentration in the wastewater was measured using the
Mg2+ 4.6 ± 0.1 standard methods for the examination of water and wastewater
Mn2+ <0.09 (APHA, 1998). Alkalinity was measured by titrating 100 mL of the
Fe (total dissolved) <0.01
Cu2+ <0.01
liquid sample with diluted sulfuric acid to pH 4.5 (APHA, 1998).
Al3+ <0.02 The total carbon (TC), total inorganic carbon (TIC) and total organic
a
carbon (TOC) concentrations were measured using a Shimadzu
after supplemented with 15% CO2 .
b
ND: not detected. TOC-VCPH analyzer (Tokyo, Japan). The solution pH, electric con-
ductivity and oxidation reduction potential were measured using
an Orion 5-Star pH/ORP/Cond./DO Meter (Thermo Scientific, USA).
removal and lipid production (Bruce, 2008; Wang et al., 2010a,b; The experiments were performed in triplicates and the average
Li et al., 2011a; Zhou et al., 2011; Abou-Shanab et al., 2013). values were reported with their standard deviations. The control
Limited numbers of studies have documented robust algal (wastewater without algal inoculums) was conducted under the
strains with consistent growth in wastewater containing low con- same conditions.
centration of nutrients and an additional CO2 amendment (Wang
et al., 2010a; Xin et al., 2010; Cho et al., 2011). In addition, there is a
2.3. Algae cultivation, growth, CO2 consumption rate, and
lack of information on the kinetic assessment of biomass and lipid
nutrient removal
production of microalgae cultivated in nutrient-limited growth
media (i.e., wastewater). Therefore, the present study focused on
The experiments were carried out in two phases. The first
the screening of potent algal strains based on the kinetic assess-
phase was conducted to select microalgae species with the high-
ment of specific growth rate and specific nutrient consumption
est growth based on their biomass yield after 7 days of cultivation
rate under nutrient-limited conditions. This study also focused on
in the tertiary wastewater effluent. The second phase focused
the application of these strains for simultaneous nutrient removal
on the determination of nutrients removal and biodiesel produc-
and lipid production from tertiary-treated municipal wastewater
tion using selected microalgal species in batch experiments. The
effluent supplemented with CO2 .
batch experiments were conducted using 500 mL aluminum crimp-
sealed serum bottles containing 200 mL filter sterilized tertiary
2. Materials and methods wastewater, inoculated with 2% (Vinoculum /Vwastewater ) and supple-
mented with 15% CO2 . The bottles were incubated in a shaker
2.1. Algal strains, culture conditions, and inoculums preparation incubator at 150 rpm, 27 ◦ C, under white fluorescent light illumi-
nation (alternate light/dark periods of 16 h/8 h) at an intensity of
Seven microalgal species were investigated in this study: 45–50 ␮mol photon m−2 s−1 for 7 days. During incubation, 6 mL of
Chlamydomonas pitschmannii GU732416, C. mexicana GU732420, C. mixed liquor was periodically collected from each serum bottle to
vulgaris GU732417, M. reisseri FR751203, C. ellipsoidea GU732422, measure T-N, T-P and TIC. The OD680 were converted to dry cell
S. obliquus FR751175 and O. multisporus GU732424 (Table 2). The weight (DCW) concentration (g L−1 ), based on a linear relationship
microalgal strains were individually inoculated in 250 mL Erlen- between the OD680 and dry cell weight (APHA, 1998), which was
meyer flasks containing 100 mL Bold’s Basal Medium (BBM) at obtained after an extensive data analysis and is given by Eqs. (1), (2)
10% concentration (Vinoculum /Vmedia ) (Bischoff and Bold, 1963). The and (3) for C. vulgaris, S. obliquus and O. multisporus, respectively,
microalgal cells were cultivated in a shaker incubator at 150 rpm as follows:
and at 27 ◦ C under continuous illumination of white fluorescent
light of 45–50 ␮mol photon m−2 s−1 for two weeks. The microalgal Dry weight (g L−1 ) = 0.3499 × OD680 − 0.0022 (R2 = 0.9982) (1)
suspension in the BBM was adjusted to an absorbance of 1.5 at an
optical density (OD) of 680 nm as measured using a spectropho-
tometer (Hach DR/4000, Loveland, Colorado, USA). The algal cells Dry weight (g L−1 ) = 0.3521 × OD680 + 0.0038 (R2 = 0.9942) (2)
were harvested by centrifugation at 3000 rpm for 15 min at 4 ◦ C
and washed with NaHCO3 solution (15 mg L−1 ). Two milliliters of
microalgae were used as initial inoculums for further experiments. Dry weight (g L−1 ) = 0.3340 × OD680 + 0.0028 (R2 = 0.9955) (3)
144 M.-K. Ji et al. / Ecological Engineering 58 (2013) 142–148

The specific growth rate () was calculated by fitting the dry cell
weight for the first 7 days of cultivation to an exponential function,
as shown in Eq. (4) (Jiang et al., 2011):
ln N2 − ln N1
= (4)
t2 − t1
where, N1 and N2 are defined as the dry cell weight at times t1
and t2 , respectively. The optimal specific growth rate (opt ) which
is essentially equal to the maximum specific growth rate (max )
was used in this study for comparison purpose with the existing
experimental results because the growth conditions employed in
this study were nutrient limited.
The initial substrate removal rate (Ri) was calculated using in
Eq. (5):
S0 − St
Ri = − (5)
t0 − tt
where, S0 is the initial substrate concentration as T-N or T-P, and
St is the corresponding substrate concentration at “tt ”. Therefore
the specific rate of substrate removal (RXi ) can be calculated as
RXi = Ri /biomass.

2.4. Lipid extraction and fatty acid profile analysis

Total lipids were extracted from freeze-dried algal biomass

using the modified method described by Bligh and Dyer (1959).
Each experiment was carried out in triplicate and the average val-
ues were reported. The fatty acids were analyzed using a method
modified by Lepage and Roy (1984). The fatty acid methyl esters
(FAMEs) were analyzed by gas chromatography with a flame
ionization detector and a HP-INNOWax capillary column (Agi-
lent Technologies, USA). Helium was used as a carrier gas at
2.2 mL min−1 . The injection volume and split ratio were 2 ␮L and
45:1, respectively. The temperature of the injector and detector Fig. 1. Variation of dry cell weights (a) and specific cell growth rates (b) according to
growth of the three microalgal species cultivated in the tertiary-treated municipal
were set at 250 and 275 ◦ C, respectively. Mix RM3, Mix RM5, GLC50
wastewater amended with 15% CO2 . Inset shows the concentration of total inorganic
and GLC70 (Supelco, USA) were used as standards. carbon remained in the wastewater after the 7-day cultivation.

3. Results and discussion

3.1. Wastewater characterization
The physicochemical properties of the filtered tertiary waste-
water were analyzed (Table 1). The wastewater used in the this
study showed a C:N:P molar ratio of 22:11:1, which indicated a
slightly high N concentration. Phang and Ong (1988) reported that
a medium with a balanced supply of N and P, and an optimal C:N:P
molar ratio equal to 56:9:1 could be suitable for benthic microal-
gae. Xin et al. (2010) concluded that the proper N:P molar ratios for
nutrient removal ranged from 11:1 to 18:1. The deficiency of car-
bon content in wastewater can result in a substantial inhibition of
algal growth and thus the incomplete removal of nutrients (Woertz
et al., 2009). Zhou et al. (2012) reported that the biomass yield (0.69
and 1.16 g L−1 ), and N (39 and 100%) and P (42 and 90%) removals
were achieved by Auxenochlorella protothecoides grown on waste-
water amended with 0.03 and 5% CO2 , respectively. Therefore, the
initial C:N:P molar ratio was adjusted to 78:11:1 in this study by
supplying 15% CO2. The total N, P and other trace nutrients (Table 1)
in wastewater supported the growth of algae.
3.2. Growth rate and biomass yield of microalgal species
The dry biomass yield of seven microalgal species cultivated
in tertiary municipal wastewater effluent without CO2 supple-
mentation were determined (Table 2). The biomass yield of O.
multisporus, C. vulgaris and S. obliquus accounted for higher dry
weight of 0.203, 0.197 and 0.197 g dwt L−1 , respectively, compared
to the other four microalgal species. These microalgal species were
isolated from nutritionally-unbalanced environments (e.g., a low
level of nutrients), and thus they adapted and grew well in the low
nutrient conditions of the tertiary effluent wastewater. The high-
est dry biomass (0.20 g dwt L−1 ) was observed for O. multisporus
grown in wastewater. This biomass was relatively low compared
to the typical yield (0.3–0.6 g L−1 ) expected in microalgae cultiva-
tion (Sydney et al., 2011) due to the low concentration of nutrients
particularly N, P and TIC in the tertiary-treated municipal waste-
water used in this study (Table 1). Nitrogen is an important macro
element contributing to biomass production (1–10% of the total
mass), and is also a critical factor in regulating algal lipid content
(Becker, 1994). Phosphorus is essential for growth and many cellu-
lar processes such as energy transfer and the biosynthesis of nucleic
acids in algal cells (Chen et al., 2011).
Microalgal species (C. vulgaris, O. multisporus and S. obliquus)
that showed higher biomass yield were selected for further inves-
tigation of their growth rates and nutrients removal efficiency from
wastewater in the presence and absence of 15% CO2 . The dry cell
weight of the three microalgal species grown in the wastewater
effluent ranged from 0.29 to 0.31 g L−1 (Fig. 1a). As appeared in
Fig. 1b, C. vulgaris showed the highest growth rate (1.37 d−1 ) after
3 days of cultivation due to the adjusted suitable C:N:P ratio. When
the N and P were depleted below a certain level (<0.1 mg L−1 ), the
algae growth rate decreased and the growth reached steady state
at 5–7 days. Nannochloropsis sp. has been reported to have a high
 M.-K. Ji et al. / Ecological Engineering 58 (2013) 142–148 145

Table 2
Dry biomass concentrations of microalgae cultivated in the tertiary-treated municipal wastewater without added carbon dioxide. The biomass weight was determined after
the 7-day cultivation.

Algal species Strain ID Sources Accession number Algal dry biomass in wastewater (g L−1 ) References

O. multisporus YSW008 Wastewater GU732424 0.203 Abou-Shanab et al. (2011b)

C. vulgaris YSW004 Wastewater GU732417 0.197 Ji et al. (2012)
S. obliquus YSR008 River FR751175 0.197 Abou-Shanab et al. (2013)
C. mexicana YSL007 Lake GU732420 0.127 Abou-Shanab et al. (2011a)
C. ellipsoidea YSR003 River GU732422 0.119 Abou-Shanab et al. (2011b)
M. reisseri YSL017 Lake FR751203 0.109 Abou-Shanab et al. (2013)
C. pitschmannii YSL003 Lake GU732416 0.103 Abou-Shanab et al. (2011a)

biomass production with a significant increase in its specific growth

rate after 2–4 days using growth medium amended with 15% CO2
(Jiang et al., 2011). Cho et al. (2011) reported that a dry weight of
0.41 g L−1 was observed in Chlorella sp. grown on secondary efflu-
ent containing 19.9 mg L−1 total nitrogen. A higher  (1.37 d−1 ) was
obtained for C. vulgaris at day 3 in comparison to previous reports
(Table 3). The total inorganic carbon (TIC) of the wastewater was
decreased from 51.7 to 9.7, 10.8 and 12.9 mg L−1 for O. multisporus,
C. vulgaris and S. obliquus, respectively. A previous study reported
the mechanism of inorganic carbon utilization in microalgae, and
that the bicarbonate ion (HCO3 − ) can serve as an alternative carbon
source for phototrophic microalgae (Huertas and Lubian, 1998).

3.3. Nutrient removal as a result of algal growth

It is important to select a potent microalgae species in order

to investigate the coupling of advanced wastewater treatment and
biodiesel production. Fig. 2 shows the removal of total nitrogen and
phosphorus from the tertiary wastewater. It was observed that the
concentrations of TN and TP decreased rapidly due to their fast
assimilation by C. vulgaris, O. multisporus and S. obliquus in the
first 3 days of cultivation, while the TN and TP were completely
removed within 4 days. The culture with a higher specific growth
rate showed better removal of TN and TP (Fig. 2a, b). Cho et al.
(2011) reported that concentrations of TN and TP in municipal
wastewater were significantly decreased from 19.1 to 1.5 mg L−1
and from 3 to 0.2 mg L−1 , respectively, after 9 days of Chlorella
sp. cultivation. C. vulgaris and S. obliquus showed higher removal
rates of TN and TP compared to O. multisporus (Fig. 2c). The maxi-
mum specific consumption rates of TN (16.8 mg-N g-cell−1 ) and TP
(3.1 mg-P g-cell−1 ) were observed with C. vulgaris and S. obliquus,
respectively. The N and P removal rates of C. vulgaris have been
reported to be 10.5 mg T-N L−1 d−1 and 2.0 mg T-P L−1 d−1 (Aslan
and Kapdan, 2006). Jimenez-Perez et al. (2004) reported substan-
tially high removal rates of TN (83 and 56.1 L−1 d−1 ) and TP (20.8
and 10.2 L−1 d−1 ) by S. intermedius and Nannochloris sp., respec-
tively.

3.4. Lipid productivity and lipid content

Lipids are substances related biosynthetically or functionally

to fatty acids and their derivatives (Chisti, 2007). In the present
study, the total lipid content/productivity of microalgal biomass
harvested after 7 days was determined (Fig. 3). The total lipid con-
tent of the three microalgae species cultivated in wastewater in
the absence of CO2 ranged from 21 ± 1.3% to 31.4 ± 2% based on
their dry biomass. Lipid content increased due to N and P deple-
tion (Fig. 3). The highest specific lipid productivity of C. vulgaris
was 0.164 ± 0.009 g L−1 . The total specific lipid productivities were Fig. 2. Microalgal uptake of total nitrogen (a) and phosphorus (b) from the
0.164 ± 0.009, 0.119 ± 0.006 and 0.148 ± 0.004 for C. vulgaris, S. tertiary-treated municipal wastewater supplemented with 15% CO2 . The specific
obliquus and O. multisporus, respectively. These values increased consumption rates (c) for total nitrogen and phosphorous were obtained after 4
days of cultivation.
slightly in the presence of CO2 compared to the total specific lipid
productivities of 0.120 ± 0.012, 0.086 ± 0.005 and 0.120 ± 0.008 in
 146
Table 3
Summary of the results obtained in this study and comparison with previously reported work.

Type of wastewater Microalgal Carbon dioxide Light intensity Cultivation Pretreatment Initial nutrient Nutrient Dry cell weight max a (day−1 ) Lipid Content References
or medium species (%) (␮mol m−2 s−1 ) period (day) (mg L−1 ) removal ratio (g L−1 ) (%)
light(h): (%)
dark(h)

Modified BG-11 Chlorella 6% CO2 47 (24:0) 10 Autoclave TN = 250 – 0.23 0.22 – Chinnasamy
vulgaris ARC 1 et al. (2009)
TP = 40 –

Modified Bristol Chlorella 6% CO2 40 (12:12) 20 Autoclave TN = 41 – 0.98 0.27 – de Morais and

M.-K. Ji et al. / Ecological Engineering 58 (2013) 142–148

kessleri Costa (2007)
TP = 53 –

Municipal Chlorella 15% CO2 45 (16:8) 7 Filtration TN = 8.7 100 0.29 1.37 b 30 This study
wastewater vulgaris w/(0.2 ␮m)
membrane
TP = 1.7 100

Modified DM Scenedesmus 10% CO2 60 (24:0) 12 Autoclave TN = 76 – 3.51 1.19 12 Ho et al. (2010)
sp. CNW-N
TP = 59 –

Modified BG-11 Scenedesmus 5% CO2 180 (24:0) 14 Autoclave TN = 250 – 1.8 0.94 16 Tang et al.
obliquus (2011)
TP = 40 –

Municipal Scenedesmus 15% CO2 45 (16:8) 7 Filtration TN = 8.7 100 0.31 1.14 b 27 This study
wastewater obliquus w/(0.2 ␮m)
membrane
TP = 1.7 100

Modified f/2-Si Nannochloropsis 0.5% CO2 96 (24:0) 48 Autoclave – – 7.0 0.95 15 Wang et al.
salina (2012)
– –

Municipal Ourococcus 15% CO2 45 (16:8) 7 Filtration TN = 8.7 100 0.31 1.00 b 31 This study
wastewater multisporus w/(0.2 ␮m)
membrane
TP = 1.7 100
a
Maximum specific growth rate, ␮max (day−1 ).
b
Optimal specific growth rate, ␮opt (day−1 ).
 M.-K. Ji et al. / Ecological Engineering 58 (2013) 142–148
Fig. 3. Specific lipid productivity and lipid content of microalgal species cultivated
in tertiary-treated municipal wastewater with and without added carbon diox-
ide. The bars show the specific lipid productivity, while circles represent the lipid
content.
the absence of CO2 due to the increase of their growth rate and lipid
content. Nutrient limitation is an environmental stress and plays
an important role in increasing the accumulation of lipids in the
algal cells (Rodolfi et al., 2009).
Therefore, nitrogen limitation can increase the lipid and TAG
content in microalgal cells. Widjaja et al. (2009) reported that the
lipid content of C. vulgaris was only 26% of the dry biomass when
a typical level of nitrogen (70 mg NO3 − L−1 ) was used, while the
lipid content increased up to 43% with the depletion of nitrogen
(<0.02 mg L−1 ). Jiang et al. (2011) also confirmed that lipid content
continually increases even after nitrogen depletion. Under environ-
mental stress, the cessation of microalgal cell division was observed
and the synthesis of CO2 was switched to lipid production for the
storage of energy which resulted in the increased lipid content per
microalgal biomass. Upon the CO2 addition, carbon incorporation is
forced and because of N deficiency, carbon skeletons are not incor-
porated into proteins to allow the cell to grow, therefore the lipid
synthesis pathway would be taken as a carbon sink. Yongmanitchai
and Ward (1991) reported that the lipid content of Phaeodactylum
tricornutum increased when the cells were cultivated under a high
CO2 (5–10%) supply.
3.5. Fatty acid composition
Fatty acid profile has been used as potential indicators of
biodiesel quality. The C16 and C18 series content (as % of the total
FAME) of microalgae have been used to evaluate the oil/biodiesel
productivity (Tang et al., 2011). Important fuel properties of
biodiesel that are influenced by the fatty acid profile and in
turn, by the structural features of various fatty esters include
cetane number (CN) and ultimately exhaust emissions, heat of
combustion, cold flow, oxidative stability, viscosity and lubricity
(Gouveia et al., 2009). The fatty acid composition of C. vulgaris, S.
obliquus and O. multisporus were determined after 7 days of cul-
tivation, and were found to be mainly composed of palmitic acid
(C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2)
and linolenic acid (C18:3), which accounted for 25–26/24–44%,
3–6/5–16%, 11–23/22–44%, 21–38/10–25% and 20–27/7–12% of the
total fatty acids in the absence and presence of CO2 , respectively.
The amount of saturated (C16:0 and C18:0), mono-unsaturated
(C18:1) and poly-unsaturated (C18:2 and C18:3) fatty acids were
determined in C. vulgaris, S. obliquus and O. multisporus which
accounted for 29/44/27%, 38/25/37% and 60/22/18%, respectively,
in presence of CO2 . The highest concentration of saturated fatty
acid (60%) was observed in O. multisporus in wastewater amended
147
Fig. 4. Fatty acid composition of microalgal species cultivated in the tertiary-treated
municipal wastewater with and without added carbon dioxide.
with CO2 . It has been reported that more saturated fatty acid
could provide biodiesel with a higher CN, decreased NOx emis-
sions, a shorter ignition delay time and oxidative stability but
poor low-temperature properties (to gel at ambient temperature).
Poly-unsaturated fatty acids produce biodiesel with good cold-flow
properties, but susceptible to oxidation (Antolin, 2002). Oils with
high mono-unsaturated (oleic acid) fatty acid content have been
reported to have a reasonable balance of fuel properties, including
ignition quality, combustion heat, cold filter plugging point (CFPP),
oxidative stability, viscosity and lubricity (Rashid et al., 2008). Oleic
acid content increased due to deficiency of N and P, and accounted
for 44, 25 and 22% of the total fatty acid content in C. vulgaris,
S. obliquus and O. multisporus, respectively (Fig. 4). Several stud-
ies have reported that N and P deficiency results in an increase in
oleic acid content in algae cells up to 0.4–12.1%, 16–31%, 4.1–16.4%
and 7.5–58% for Dunaliella tertiolecta, C. vulgaris, Nannochloropsis
sp. and Coccomyxa sp., respectively (Hu and Gao, 2006; Ho et al.,
2010; Msanne et al., 2012). The accumulation of oleic acid paral-
lel to the reduction of cell division observed in nutrient-deficient
medium seems to confirm the key function that oleic fatty acid
plays in the processes of microalgal cell division (Siron et al., 1989),
since the desaturation of oleic acid requires oxygen and essentially
takes place inside chloroplasts. The accumulation observed in this
study might be the result of an alteration of the photosynthetic
assimilation processes in C. vulgaris, S. obliquus and O. multisporus.
In addition, the fatty acid methyl esters (FAMEs) of C. vulgaris were
composed of more unsaturated fats (18:1, 18:2 and 18:3), which
are more suitable for use in cold weather environments due to their
typically lower gel point (Belarbi et al., 2000).
4. Conclusions
Tertiary-treated domestic wastewater amended with 15% CO2
resulted in significant increases in the growth rate, biomass yield
and lipid productivity of microalgal species (O. multisporus, C. vul-
garis and S. obliquus). C. vulgaris showed the highest specific growth
rate (1.37 day−1 ), specific lipid productivity (0.164 g-lipids g-cell−1
day−1 ) and oleic acid content (44%). The concentrations of TN
and TP were decreased below their respective detection limits
within 4 days of cultivation, which was caused by an increase in
lipid and oleic acid accumulation in the microalgae. This study
demonstrated a cost-effective and environmentally sustainable
method for tertiary wastewater treatment and algal biomass pro-
duction.
148 M.-K. Ji et al. / Ecological Engineering 58 (2013) 142–148
Acknowledgements
This work was supported by the Senior Researchers program
(the National Research Foundation of Korea, 2010-0026904), the
Eco-Innovation project (Global-Top project) of the Korea Ministry
of Environment, and the Brain Korea-21 (BK-21) program of the
Korea Ministry of Education, Science & Technology (MEST).
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