Environ Sci Pollut Res (2017) 24:25618–25626
DOI 10.1007/s11356-016-7013-6
4TH INTERNATIONAL SYMPOSIUM ON ENVIRONMENTAL BIOTECHNOLOGY AND ENGINEERING-2014
Biomass and lipid production from Nannochloropsis oculata
growth in raceway ponds operated in sequential batch mode
under greenhouse conditions
Aarón Millán-Oropeza 1 & Luis Fernández-Linares 1
Received: 13 January 2016 / Accepted: 1 June 2016 / Published online: 6 June 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract The effect of sequential batch cultures of the marine
microalgae Nannochloropsis oculata on lipid and biomass
production was studied in 200-L raceway ponds for 167 days
(nine harvesting cycles) during winter and spring seasons un-
der greenhouse conditions. The highest biomass concentration
and productivity were 1.2 g/L and 49.8 mg/L/day on days 73
(5th cycle) and 167 (9th cycle), respectively. The overall in-
terval of lipid production was between 131 and 530 mg/L.
Despite the daily and seasonal variations of light irradiance
(0–1099 μmol photon/m2 s), greenhouse temperature (2.1–
50.7 °C), and culture temperature (12.5–31.4 °C), ANOVA
analysis showed no statistical difference (p value > 0.01) on
the fatty acid methyl ester (FAMES) composition over the
nine harvesting cycles evaluated. The most abundant
FAMES were palmitic (C16:0), stearic (C18:0) and
palmitoleic (C16:1Δ9) acids with 37.1, 28.6, and 8.4 %, re-
spectively. The sequential batch cultures of N. oculata in race-
way ponds showed an increasing biomass production in each
new cycle while keeping the quality of the fatty acid mixture
under daily and seasonal variations of light irradiance and
temperature.
Keywords Biofuels . Sequential batch culture . Lipids .
Microalgae . Nannochloropsis oculata . Raceway ponds
Responsible editor: Gerald Thouand
* Luis Fernández-Linares
lfernand36@gmail.com
1
Departamento de Bioprocesos, Unidad Profesional Interdisciplinaria
de Biotecnología - Instituto Politécnico Nacional (UPIBI - IPN), Av.
Acueducto s/n Col. Barrio la Laguna Ticomán, 07340 Mexico
City, Mexico
Abbreviations
DCW Dry cell weight
FAMES Fatty acid methyl esters
SBC Sequential batch culture
Introduction
Microalgae have emerged in the last decade as a promising
biotechnological option to face the fossil fuel scarcity (Chisti
2007) that is expected to occur in the second half of the present
century (Capellán-Pérez et al. 2014). In this regard,
microalgae have the ability to synthesize precursor molecules
for the production of renewable fuels like bioethanol,
biohydrogen, biomethane, or biodiesel (Singh and Gu 2010).
Microalgae can be grown in open ponds or photobioreactors.
The use of open systems to produce biofuel feedstock is 2.5
times less expensive than using photobioreactors (Richardson
et al. 2012). For that reason, open systems are currently the
preferred option for large-scale bioprocessing. However, ef-
forts to prevent contamination and control the culture condi-
tions have been very challenging. The temperature and light
irradiance variations are practically impossible to be con-
trolled in outdoor cultures (Borowitzka and Moheimani
2013). Such fluctuations can affect microalgal growth at
the point to collapse the whole culture (Mata et al. 2010).
Furthermore, contamination by predators, protozoa, ciliates,
and other fast-growing heterotrophic bacteria have restricted
the commercial production of algae in open systems to only
those organisms that can grow under specific conditions such
as high pH (Pawlowski et al. 2015) and hypersaline cultures
(Das et al. 2011) or where suitable irradiance is available
(Boruff et al. 2015). For all these reasons, a crucial factor
for the success in culturing open systems is to choose a
microalgal strain with the ability to grow in a selective
Environ Sci Pollut Res (2017) 24:25618–25626
media, as well as under daily temperature and irradiance
fluctuations without compromising the yield and quality of
the biomass.
Another point to take into account is the operational mode
of the open systems. The main cultivation modes are in batch,
feed-batch, continuous, and sequential batch culture (SBC).
The SBC consists of periodic withdrawals of volume from
the culture and the removed volume is restituted with fresh
medium. This operational mode offers several advantages for
long-term outdoor cultures because it (a) allows maintaining
log phase growth, (b) avoids the shading effect caused by high
cell density (Ho et al. 2014), and (c) allows inoculum and
biomass availability during each harvesting cycle (Fábregas
et al. 1995).
SBC cultures have been studied for wastewater removal
(Zhou et al. 2012), CO2 fixation (Bao et al. 2012; Ho et al.
2012), and enhancement of biomass productivity in several
microalgae species (Chiu et al. 2009; Ho et al. 2014;
Radmann et al. 2007), but little work has been published on
long-term pilot-scale open systems concerning its effect on the
fatty acid methyl ester (FAMES) content, which are the feed-
stock for microbial biodiesel production.
In this work, the marine microalgae Nannochloropsis
oculata was grown in 200-L raceway ponds under greenhouse
conditions and was used to study the effect of SBC during
winter and spring seasons on lipid production, fatty acid com-
position, and biomass production.
Materials and methods
The effect of SBC of the marine microalgae N. oculata on
lipid and biomass production was studied in 200-L raceway
ponds for 167 days (9 harvesting cycles) during winter and
spring seasons under greenhouse conditions. Over the SBC,
we monitored biomass concentration, lipid production, pig-
ment content (chlorophyll a and b and carotenoids), nitrate
concentration in the medium, light irradiance, culture temper-
ature, and greenhouse temperature as described below.
Microalgae cultures
The microalgae N. oculata was obtained from the Centro de
Investigación Científica y de Educación Superior de Ensenada
(CICESE, Mexico) collection. Cells were cultivated in modi-
fied f/2 medium with artificial sea water (Chiu et al. 2009)
using the following composition (g/L): 29.23, NaCl; 1.1,
KCl; 2.45, MgSO4 7H2O; 1.83, CaCl2 2H2O; 0.25, NaNO3;
0.25, NaHCO3; and 3 ml of trace element (TE) solution. The
TE solution contained the following (g/L): 5, NaH2PO4 H2O;
4.1, Na2EDTA; 3.16, FeCl3 6H2O; 0.18, MnCl2 4H2O; 0.01,
CoCl2 6H2O; 0.0, CuSO4 5H2O; 0.023, ZnSO4 7H2O; and
0.006, Na2MoO4. The inoculum was obtained from cells
25619
cultured in 10-L cylindrical plastic photobioreactors at 25
± 1 °C under photoperiods of 12:12 h (light/dark) using
cool-white fluorescent light at intensity of 100 μmol photon/
m2/s. The inoculums were aerated at a constant rate of 10 L of
air/min. Two raceway ponds of 200 L were inoculated by
adjusting the initial cell concentration to approximately
6 × 106 cells/mL and simultaneously operated over the whole
study. The experiments were conducted under greenhouse
conditions in Mexico City, Mexico (19.513° N, 99.126° W).
Raceway ponds
The open channel raceway reactor (Fig. 1) used for cultivation
consisted of two 2.05-m channels, each of 0.255 m wide and
connected by 180° bends at both ends to give a total surface
area of 1.2 m2. The raceway was constructed with fiberglass
and the culture was circulated and mixed by a four-paddle
wheel system at a superficial flow velocity of 0.30 m/s. The
operation volume was 200 L, with a culture medium depth of
0.25 m. The paddle wheel was driven by a 0.5-HP geared
electric motor Baldor brand. The cultures were conducted in
a greenhouse (under diurnal and seasonal cycles). Light irra-
diance, culture temperature, and greenhouse temperature were
recorded with a data logger T&D RTR-500. The harvesting
cycles were carried out before nitrates were completely con-
sumed (<10 mg/L, concentrations were determined as de-
scribed below); to do so, half of the culture volume (100 L)
was removed and replaced with the same volume of fresh
medium. Furthermore, at the beginning of each harvesting
cycle, additional NaNO3 was supplied to adjust the initial
nitrate concentration to 250 mg/L in order to avoid nitrate
starvation during the cultures. During the winter period, a
water heater of 200 W was used to reduce the temperature
decrease in the cultures; previous studies (data not presented)
showed that the addition of heat is crucial to avoid the collapse
of the culture in the winter seasons. Water losses due to evap-
oration were replaced periodically.
Nitrate quantification
The nitrate concentration in the medium was measured ac-
cording to a modified version of the Keeney-Nelson method
(Keeney and Nelson 1982). Culture samples of 2 mL were
centrifuged at 17,000×g for 5 min; 500 μL of the supernatant
was transferred into glass tubes dried in a dry bath block
(Major Science, USA). Then, 500 μL of 2-4 biphenyl sulfonic
acid was added to the dried samples, followed by gentle addi-
tion of 2.2 mL of KOH 12 N. The reaction was allowed to
occur for 5 min and 100 μL of the resulting supernatant was
placed in plastic cuvettes containing 900 μL of deionized
water. Optical density (OD) was measured at 410 nm. The
nitrate concentration was calculated from a standard curve of
NaNO3 ranging from 0 to 500 mg NO3/L (R2 = 0.998).
25620
Fig. 1 Raceway open pond dimensions: a top and b front view. Numbers are expressed in centimeters
Biomass determination
Dry cell weight (DCW) was determined from 5-mL culture
samples filtered in preweighed 0.7-μm glass-fiber filters, the
excess of salts was removed with 15 mL of NH4HCO3 0.5 N
(Zhu and Lee 1997), and the filters were dried completely after
24 h of oven incubation at 60 °C and then weighed (measure-
ment error = 0.002 mg). OD was measured at 600 and 750 nm.
Direct cell count was done by using a counting Neubauer
hemocytometer, a Swift M10L digital microscope, and the
software Cell C Counting (Selinummi et al. 2005). Each mea-
surement was performed in three technical replicates.
The specific growth rate (μ) corresponding to the exponen-
tial growth phase was calculated according to
μ ¼ ln ðX 1 − X 0 Þ = t 1 −t 0
where X1 represents the dry biomass (g/L) at time t1 (h), and
X0 represents the initial concentration (g/L) at time t0 (h).
Environ Sci Pollut Res (2017) 24:25618–25626
Lipid extraction
Lipid extraction was performed using n-hexane due to its
lower toxicity compared to other chloroform/methanol-
based extraction protocols. Culture samples of 30 mL
were concentrated by centrifugation (4300×g/15 min),
the pellet was washed with 20 mL of deionized water
and centrifuged again (4300×g/15 min), and the biomass
was dried at 60 °C. Dried biomass was milled and resus-
pended in 4 mL of n-hexane. The suspensions were son-
icated for 30 min at 30 kHz and stored for 12 h at 5 °C.
The lipid extract was transferred to a preweighed vial;
two washes of the residual biomass were performed by
adding 3 mL of n-hexane and the solvent was recovered
with the initial extract (10 mL of final lipid extract). The
ð1Þ n-hexane was removed with nitrogen gas and the vials
were dried until constant weight and finally weighed.
Each measurement was performed in three technical
replicates.
Environ Sci Pollut Res (2017) 24:25618–25626
Fatty acids composition analysis
Lipid extract was transesterified with 750 μL of methanolic
chlorhydric acid 0.5 M at 80 °C for 3 h. FAMES were dis-
solved in 1 mL of hexane and filtered with a 0.2-μm
polytetrafluoroethylene membrane. Samples of 2 μL of
FAMES were analyzed by gas chromatography (GC) on a
Clarus 500 gas chromatograph (Perkin Elmer). A 30-m col-
umn was used for the separation (AT-WAX, polyethylene gly-
col with 0.25 mm internal diameter and 0.2-μm film thick-
ness). Helium was used as carrier gas at 0.96 mL/min flow
rate. The injector temperature was set at 230 °C and the de-
tector temperature (flame ionization detector) was set at
250 °C. The oven temperature was programmed to start and
stay at 140 °C for 5 min, then from 140 °C to 240 °C at
10 °C/min, and maintained 15 min at 240 °C. Retention times
of the components were taken from a standard that was used
as reference (Supelco 37 FAME mix, Sigma Aldrich No.
47885-U) to identify the FAMES in the samples. The
FAMES standard reference was verified by GC-MS. The mass
spectrometer (MS), transfer line, and ion source quadrupole
temperatures were 200, 230, and 150 °C, respectively. The
MS was operated at electron ionization positive mode (70 eV).
Pigment content
Carotenoids and chlorophylls were measured according to the
protocols and the equations obtained by Ritchie (2006) and
Strickland and Parsons (1972). Culture samples of 1.5 mL
were centrifuged for 5 min at 17,000×g, after which the su-
pernatant was removed. The cells were suspended in 1.5 mL
of methanol and incubated at 45 °C in the dark for 30 min. The
tubes were centrifuged (17,000×g/5 min) and the methanolic
extract was transferred into plastic cuvettes for measurement
at 480, 652, 665, and 750 nm. Carotenoids and chlorophyll A
and B concentrations were estimated according to the follow-
ing equations:
Chlorophyll‐A ½mg=mL ¼ −8:10 OD652nm ð2Þ
þ 16:57 OD665nm −OD750nm
Chlorophyll‐B ½mg=mL ¼ 27:44 OD652nm ð3Þ
−12:17 OD665nm −OD750nm
Carotenoids ½mg=mL ¼ 4OD480nm −OD750nm ð4Þ
Statistical analysis
The extracted FAMES, pigments, and biomass productivities
from N. oculata were compared according to the values ob-
tained in the harvesting cycles during spring and winter sea-
sons using one-way ANOVA and Tukey test. The level of
significant difference was set at p value <0.01.
25621
Results and discussion
Effect of sequential batch culture on cell growth
The SBC culture of N. oculata was carried out in raceway
ponds of 200 L for 89 days in winter and 46 days in spring
(Fig. 2a). This type of culture allowed harvesting portions of
100 L in each harvesting cycle and using the remaining 100 L
as inoculum for the next culture cycle. The sequential batches
lasted 8 to 19 days, a period in which a change of pH was
observed, from pH = 8 at the beginning of each cycle to
pH = 10 at the end (data not shown). The high pH of the
medium was a crucial factor to keep monoalgal cultures and
to avoid contamination from predators. The highest biomass
concentration was 1.2 g/L and it was achieved at the end of the
5th cycle (73 days of culturing).
It is widely known that nitrogen deprivation decreases cell
growth in microalgal cultures (Chen et al. 2011; Rodolfi et al.
2009). In order to ensure nitrogen availability for each har-
vesting cycle during the entire culture period, the fresh medi-
um was supplemented with NaNO3.
Even if nitrate depletion was observed only in the second
culture cycle, between 19 and 27 days of culture (Fig. 2b), the
biomass productivity in the second culture cycle did not de-
crease in comparison with the previous cycle (Fig. 3). This can
be attributed to the immediate change of medium that was
carried out on day 27 to avoid both, long period of nitrogen
limitation and cell growth decrease.
Furthermore, DCW (g/L) and direct cell counting (106
cell/mL) showed a Pearson correlation coefficient of
R2 = 0.96 (Fig. 4). This highly linear value is consistent with
the measured biomass belonging to N. oculata rather than to
other microorganisms in the non-axenic open culture.
Effect of diurnal and seasonal fluctuations of irradiance
and temperature on cell growth and lipid production
Over the whole culture period, the interval of maximum and
minimum biomass productivities was between 24.4 ± 0.6 and
49.8 ± 2.1 mg/L/day. Taking into account each harvesting cy-
cle, the average biomass concentrations were 667 ± 302 and
624 ± 85 mg/L during winter and spring seasons, respectively.
These intervals of biomass productivities and concentrations
can be attributed to the dynamic conditions of light irradiance
and temperature. The greenhouse temperature intervals were
between 2–47 °C and 8–51 °C in the winter and spring, re-
spectively (Fig. 5a, b). The temperature intervals inside the
raceway cultures were in the range of 8–31 °C in the winter
and 8–26 °C in the spring (Fig. 5c, d). Regarding the avail-
ability of light or irradiation, these are distributed according to
daily circadian cycle where the average ratios of light/dark
hours per day were 10:14 and 12:12 for winter and spring
25622 Environ Sci Pollut Res (2017) 24:25618–25626

Fig. 2 Sequential batch culture

of N. oculata in 200-L raceway
ponds. a Biomass concentration
and b nitrate concentration in the
medium. Dashed lines indicate
the harvesting cycles for the
winter (white circle, white
triangle) and spring cultures
(black circle, black triangle). Data
are the mean ± standard deviation
(n = 2)

respectively, and the maximum radiations were obtained be-
tween noon and 14 h (Fig. 5e, f).
In a previous study, the optimal laboratory conditions for
the cell growth of N. oculata in 2.5-L bubble column
photobioreactors were 21 °C, pH = 8.4, and light irradiance
of 52 μmol photon/m2/s (Spolaore et al. 2006) while using a
medium with a formulation similar to the one used in this
work. Compared to that work, the raceway cultures in this
study were under suboptimal conditions of temperature and
light intensity during most part of the time. At this respect, the
average temperatures inside the raceways cultures were 19.7
± 5.5 and 18.1 ± 4.8 °C in winter and spring, respectively. The

Fig. 3 Biomass productivities of the seasonal (winter/spring) harvesting Fig. 4 Pearson correlation between DCW (g/L) and direct cell count
times during the sequential batch culture of N. oculata in 200-L raceway (106 cell/mL) of the sequential batch culture of N. oculata in 200-L
ponds. Data are the mean ± standard deviation (n = 2) raceway ponds
Environ Sci Pollut Res (2017) 24:25618–25626
Fig. 5 Variation of the greenhouse temperature (a, b), raceways culture
temperature (c, d) and light irradiance (e, f) over the sequential batch
culture of N. oculata in 200-L raceway ponds in the winter (a, c, e) and
intervals of maximum growth rates were 0.002–0.0136 h−1 for
winter and 0.0027–0.0062 h−1 for spring; additionally, no sta-
tistical difference was observed between the studied periods
(p > 0.01). The highest growth rate obtained in this study
(0.0136 h−1) was 2.6 times lower than the one reported by
Spolaore et al. (2006) that is 0.0359 h−1. The latter can be
related to the fact that outdoor cultures are susceptible to con-
stant temperature and irradiation fluctuations (Fig. 5), making
it impossible to control the optimal conditions. The outdoor
production of microalgae is restricted by light limitation not
only at the beginning and end of the day but also during the
night period. In addition, solar irradiance also varies through-
out the year and outdoor cultivation clearly leads to a more
complex operation process than during continuous cultivation
based on artificial light.
In this work, light irradiance reached a maximum value of
1099 μmol photon/m2/s during the spring season or period
(Fig. 5f). Compared to the optimal irradiance reported for
N. oculata in laboratory conditions, 52 μmol photon/m2/s
(Spolaore et al. 2006), the raceway cultures of this work were
exposed to high levels of light irradiance, 9 and 10 h per day
25623
spring (b, d, f) seasons. Each boxplot contains all the recorded values for
the specific day hour throughout the study (n = 89 for winter and n = 47
for spring)
during winter and spring respectively. It was reported that high
levels of light intensity can collapse the outdoor cultures due
to a photoinhibitory response (Borowitzka and Moheimani
2013). In this regard, the photooxidative state in microalgae
can be estimated as a function of the carotenoids/chlorophyll
ratio (Solovchenko et al. 2009), where a value closer to 1
represents more physiological stress. This assumption is based
on the fact that the carotenoid content increases to protect the
cell from light damage, as well as that the chlorophyll content
decreases in order to recover nitrogen from chloroplasts by
promoting their lysis when nitrogen limitation occurs
(García-Ferris et al. 1996). Throughout the whole culture pe-
riod, the interval of carotenoids/chlorophyll a ratio was be-
tween 0.07 and 0.4. Even if the maximum incident irradiance
was 21 times the optimal reported value (Spolaore et al. 2006),
no decrease in cell growth was observed (Fig. 2a). However,
in the case of Scenedesmus obtusiusculus cultivated under
controlled conditions in a flat-panel photobioreactor, the
highest values of photosynthetic activity (160 mgO2/ gbiomass/h)
and biomass productivity (0.97 gbiomass/L/day) were obtained
at 620 μmol photon/m2/s, 35 °C and pH of 8 (Cabello et al.
25624 Environ Sci Pollut Res (2017) 24:25618–25626

Table 1 Lipid accumulation, pigment production, and FAMES profile overall photosynthetic efficiency (Kliphuis et al. 2011) and/or
of N. oculata over the sequential batch culture
reduction of inhibition effect due to high radiation.
Compound Winter 07/December/ Spring 12/April/ The interval of lipid concentration was from 131 ± 2 to 530
2012–08/March/ 2013–28/May/ ± 33 mg/L, which represented 26 ± 1 and 44 ± 4 % of DCW
2013 2013 lipid content, respectively (Table 1). It was reported for
Lipid production [mg/L] 131 ± 2–530 ± 33 306 ± 42–400 ± 64
N. oculata that the temperatures below the optimal (21 °C)
Lipid content [% of DCW] 26 ± 1–44 ± 4 42 ± 5–44 ± 7
reduces dramatically the growth rate (Converti et al. 2009). In
this work, the culture temperatures were below 21 °C from 4
Chlorophyll a [mg/L]* 4.9 ± 0.1–11.6 ± 1.1 3.3 ± 0.5–6.2 ± 0.6
to 11 a.m. (Fig. 5c, d), but the increasing tendency of biomass
Carotenoids [mg/L]* 0.8 ± 0.01–2 ± 0.2 0.5 ± 0.1–1.1 ± 0.1
productivity in each batch highlights the adaptability of
FAMES [% abundance]
N. oculata to grow under a wide and dynamic interval of
C14:0* 3 ± 0.4 1.8 ± 0.4
temperatures.
C16:0 37.2 ± 5.6 36.9 ± 0.3
Further ANOVA did not show statistical difference
C16:1 Δ9 9.8 ± 5.6 5.5 ± 2.4
(p > 0.01) in FAMES composition over the SBC culture
C18:0 27.1 ± 5 31.6 ± 4.1
(Table 1), except for myristic acid (C14:0) that was 1.6-fold
C18:1 Δ9 2.8 ± 1.1 2.1 ± 0.2
more abundant during winter. The most abundant FAMES
C18:3 Δ9,12,15 2.8 ± 1.9 2.2 ± 1.3
were palmitic acid (C16:0), stearic acid (C18:0), and
C20:3 Δ11,14,17 0.8 ± 0.5 0.9 ± 0.05
palmitoleic acid (C16:1 Δ9) with 37.1 ± 4, 28.6 ± 5, and 8.4
C20:5 Δ5,8,11,14,17 3.3 ± 1.9 2.8 ± 0.8
± 5 %, respectively. In several studies, C18:0 is usually found
Data are the mean ± standard deviation (n = sequential batches during the in low concentrations in microalgae and C16:0, C18:1, and
winter and spring cultures) C18:3 are the major fatty acids (He et al. 2015; Toledo-
*Statistical difference (p < 0.01) was found through the SBC Cervantes et al. 2013; Liu et al. 2011). However, Suh et al.
(2015) reported that N. oculata had a very high content of
2015). Moreover, the immersion of cells into a dark zone C18:0 (35.5 %) and C18:3n-3 (29.1 %). In the present work,
reduces photo damage effect by avoiding long exposure to the chlorophyll a and carotenoid production was 1.4- to 1.8-
high irradiance in terms of photon flux density and providing fold higher in winter than in spring showing a statistical dif-
dark time for microalgae to repair photo-induced damage ference between seasons (p < 0.01). In another study,
(Merchuk et al. 1998). Janssen et al. (2000) have reported Chlorella sp. L1 and Monoraphidium dybowskii Y2 were
that light/dark cycles in the range of 10–100 s could have a grown at different light intensities (40, 200, 400 μmol pho-
considerable influence on the efficiency of light utilization. ton/m2/s), under the higher level of light (400 μmol photon/
The light/dark cycle ratio can occur by the self-shading and m2/s) chlorophylls, were degraded while protein and carbo-
influences the biomass and byproduct productivities. hydrate content decreased; however, the accumulation of lipid
Moreover, mutual shading of the cells will result in a dark was stimulated (He et al. 2015). Coupled with the lipid accu-
zone inside the culture leading to two possible effects: lower mulation quantum efficiently (Fv/Fm) decreased under high

Table 2 Comparison of biomass and lipid productivities in open raceway ponds operated on sequential batch culture

Microalgae Raceway Location Biomass productivity Lipid productivity Reference

characteristics* [g/L/day] [mg/L/day]

Tetraselmis sp. MUR-233 A=1 Perth, Australia 0.015 N. A. Raes et al. (2014)
S = 0.22
Nannochloropsis oculata A = 1.25 Mexico City, Mexico 0.049 24.9 This study
V = 200
S = 0.30
Chlorophyta sp. A = 0.98 Sendai City, Japan 0.064 N. A. Hase et al. (2000)
V = 200
S = 0.2
Anabaena sp. ATCC 33047 A=1 Sevilla, Spain 0.10 N. A. Moreno et al. (2003)
S = 0.35
Chlorella sp. A = 0.98 Sendai City, Japan 0.103 N. A. Hase et al. (2000)
V = 200
S = 0.2
Pleurochrysis carterae A=1 Perth, Australia 0.23 78.2 Moheimani and Borowitzka
S = 0.22 (2006)

A = superficial area [m2 ], V = operational volume [L], S = velocity of the paddle wheels [m/s]
Environ Sci Pollut Res (2017) 24:25618–25626
light exposure, which dropped from 0.75 to 0.51 in Chlorella
sp. and 0.6 in M. dybowskii Y2 (He et al. 2015). Lipid accu-
mulation affected by light stress was closely related with Fv/
Fm and the changes of reactive oxygen species (Converti et al.
2009; Gwak et al. 2014).
The maximum biomass areal yield obtained in this work
was 8.3 g biomass/m2/day. Compared to other outdoor cul-
tures, it represents 12 % out of the highest values achieved in a
CBC culture of Spirulina in the 10 m3 Mangueira Lagoon in
Brazil (Morais et al. 2009) and 70 % of the Phaeodactylum
tricornutum cultures in a 0.078-m3 circular pond that was
operated in Italy (Silva Benavides et al. 2013). Regarding
the lipid accumulation in the same studies mentioned above,
the highest lipid productions for Spirulina and P. tricornutum
were only 7 and 31 % of the one achieved in the present study
(530 ± 33 mg/L), respectively.
Additionally, a comparative analysis of biomass productiv-
ities between this work and other studies carried out in race-
way systems using SBC mode is shown in Table 2. These
results showed that there is a high variability of biomass yields
among the microalgae species that depends on the geograph-
ical location, where the climatic conditions and light availabil-
ity have to be taken into account for further development of
microalgae-based bioprocesses.
The oleaginous nature of N. oculata and the invariable
FAMES composition through the daily and seasonal fluctua-
tions of light irradiance and temperature suggest that this
microalga is a suitable biological system for production of
microbial biodiesel feedstock.
Conclusion
The SBC culture of N. oculata in 200-L raceway ponds
showed an increasing biomass production in each new cycle
while keeping monoalgal cultures for 167 days. The average
biomass concentrations were 667 ± 302 and 624 ± 85 mg/L
during winter and spring seasons, respectively. The highest
biomass concentration and productivity were 1.2 g/L and
49.8 mg/L/day. The highest lipid productions were 530 ± 33
and 400 ± 64 mg/L for winter and spring seasons, respectively.
Despite the daily and seasonal variations of light irradiance
and temperature in greenhouse conditions, the fatty acid com-
position of N. oculata was stable over the culture. The most
abundant FAMES were palmitic (C16:0), stearic (C18:0), and
palmitoleic (C16:1Δ9) acids with 37.1, 28.6, and 8.4 %, re-
spectively. These results encourage further studies on
bioprocess development at larger scale using the SBC as op-
erational strategy for industrial purposes.
Acknowledgments The authors are grateful for the financial support
provided by the National Science and Technology Council (CONACYT)
of Mexico, the Secretary for Research and Postgraduated Studies—
25625
Instituto Politécnico Nacional (SIP-IPN, grants 20130388 and
20144620), and the collaboration of Research and Development Center
of CARSO. The authors would also like to thank Claudia Guerrero
Barajas and Hervé Marie for their participation in reviewing the paper.
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