Algal Research 34 (2018) 175–181

Contents lists available at ScienceDirect

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

Milking exopolysaccharides from Botryococcus braunii CCALA778 by T

membrane filtration
⁎
Rafael García-Cuberoa, , Weiliang Wangb, Judit Martínc, Elisabeth Bermejod, Lolke Sijtsmaa,
Arnoud Togtemaa, María J. Barbosae,f, Dorinde M.M. Kleinegrisa,g
a
Wageningen Food & Biobased Research, Wageningen, the Netherlands
b
Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
c
Universidad de Valladolid, Valladolid, Spain
d
Universidad de Huelva, Spain
e
Wageningen University, Bioprocess Engineering, AlgaePARC, PO Box 16, 6700 AA, Wageningen, the Netherlands
f
University of Bergen, Department of Biology, PO Box 7803, 5006 Bergen, Norway
g
Uni Research Environment, P.B. 7810, 5020 Bergen, Norway

A B S T R A C T

The aim of this work was to optimize the efficiency of extraction and recovery, also known as ‘milking’, of exopolysaccharides (excreted polysaccharides, EPS) from
continuous cultures of Botryococcus braunii CCALA778. First, an indoor process was developed and optimised, ensuring the highest milking efficiency without
compromising culture viability. For this, photobioreactors were operated in a photo-chemostat mode under simulated outdoor conditions of a typical summer at
AlgaePARC (51°59′44.1”N 5°39′26.2″E) in Wageningen, The Netherlands. Once a steady state was reached, areal productivities of 23 g m−2 d−1 and 3 g m−2 d−1 for
biomass and EPS were achieved. EPS milking was done by membrane filtration of one reactor volume at the beginning of the dark period. After optimization, the
maximum recovery of EPS, without damaging the cells, was 12%; yielding a daily EPS extraction rate of 0.36 g m−2 d−1. The optimised process was scaled-up and
applied outdoors during the summer (at AlgaePARC facilities). Outdoor cultures showed 25% lower biomass productivity (17 g m−2 d−1) but an 25% higher EPS
productivity (4 g m−2 d−1). The efficiency in the milking, however, decreased as compared to indoor results. Only 3% of the total content of EPS produced outdoors
was milked (0.12 g m−2 d−1). To improve the EPS milking process, future research should focus on increasing the EPS extraction yield without negatively influencing
its production by Botryococcus braunii.

1. Introduction scaffolds for polyesters production [10]. Moreover, based on their

Much has been written about the potential role of the colonial mi-
croalgae Botryococcus braunii in a bio-based economy [1]. This micro-
algal species displays the attractive feature of naturally excreting hy-
drocarbons and sugars that may remain attached to the colonies
forming a halo [2]. Botryococcus braunii strains can be classified into 4
different races according to the chemical profile of the excreted hy-
drocarbons: A (C25-C31), B (CnH2n–10, n = 30–37), L (C40H78 or tetra-
terpene), and S (C18-C20) [3]. Although this microalga exhibits a low
growth rate, efforts have been made to use the chemicals present in the
halo for industrial purposes [4]. The main efforts have been focused on
the microalgal production and continuous in situ extraction (also known
as ‘milking’ [5]) of hydrocarbons in B.braunii cultures [6, 7]; and less on
the use of this microalga for carbohydrates production. It has been
reported that the strain Botryococcus braunii CCALA778 (race A) ex-
cretes mainly exopolysaccharides rich in galactose and fucose [8, 9].
These monosaccharides could be used in the chemical industry as
ability to alter rheological characteristics of solutions, microalgal exo-
polysaccharides can be commercially interesting. The pseudo-plastic
behaviour of B. braunii exopolysaccharides, for instance, could have an
application in wall paints or textiles, laundry products, adhesives,
paper, and foods [2]. However, to make B.braunii production feasible
and commercially competitive, several bottlenecks should be overcome
[11]. Among them, it is necessary to ensure a continuous production
from steady cultures and to find an extraction process that provides
high yield of product without affecting the cell viability [12]. Specifi-
cally, the first bottleneck might be addressed by simulating at lab scale
the climate conditions found outdoors to check the microalgal perfor-
mance under those conditions in a well-controlled regime. These find-
ings might be applied later to check the sustainability of the EPS pro-
duction at large-scale [9]. So far, most research has been focussing on
extraction of hydrocarbons from B.braunii by organic solvents [5, 13];
supercritical CO2 [14], and less on mechanical extractions [15]. In any
case, there is a lack of knowledge on how to milk EPS from B.braunii.

⁎
Corresponding author.
E-mail address: cubero@ual.es (R. García-Cubero).

https://doi.org/10.1016/j.algal.2018.07.018
Received 30 January 2018; Received in revised form 17 July 2018; Accepted 27 July 2018
2211-9264/ © 2018 Published by Elsevier B.V.
R. García-Cubero et al.
In this work, we developed and optimised a process for solvent free,
continuous extraction and recovery of exopolysaccharides (EPS)
without affecting cell viability of Botryococcus braunii CCALA778. This
work was done under the climate conditions found during the summer
in AlgaePARC (Wageningen, The Netherlands).
2. Material and methods
2.1. Growth medium and biologic material
Botryococcus braunii CCALA 778 (non-axenic strain provided by
Culture Collection of Autotrophic Organisms. Institute of Botany CAS,
Trěboň, Czech Republic) was grown photo-autotrophically on modified
Chu 13 medium (CaCl2∙2H2O 734 μM; MgSO4∙7H2O 811 μM; K2HPO4
602 μM; KNO3 3.956 mM; FeNaEDTA 55 μM; HEPES 50 μM; Vitamin
B12 0.1 μM; Thiamine 3.26 μM; Biotin 0.11 μM; final pH 7.2).
2.2. Indoors continuous cultivations
Indoors cultivations were performed in a flat panel photobioreactor
with a light path of 0.02 m, working volume of 1.9 L and an illuminated
surface of 0.08 m2 (Labfors®, Infors, HT,2010) in photo-chemostat
mode (D = 0.25d−1) during the light hours of the day cycle
(12Light:12Dark, from 7 am to 7 pm). The bioreactors were illuminated
by a 260 LED lamps panel, with a warm-white spectrum located on one
side of the reactor. Light intensity simulated the average outdoors light
regime of 3 summer months (June, July, and August) for the last ten
years at AlgaePARC (51°59′44.1”N 5°39′26.2″E) using the European
Photovoltaic Geographical Information System (PVGIS) (http://re.jrc.
ec.europa.eu/pvgis/). Light intensity followed a sine curve during the
light period, displaying a maximum of 1100μmolph m−2 s−1 at noon.
Air was supplied at a flow rate of 0.5vvm to mix the cultures; pH was
controlled by CO2 injection on demand (pH-set point 7.2). The tem-
perature was kept at 26 °C by water recirculation through a water jacket
in direct contact with the cultivation chamber. A condenser (tempera-
ture 2 °C) was used to prevent water losses due to evaporation [16].
Steady state was assumed (μ = D) after harvesting 5 times the reactor's
volume (9.5 L) and measuring ten constant determinations of dry
weight. Once a steady state was achieved, at the beginning of the dark
period, the inlet of fresh medium stopped (dilution pumps turned off),
and milking of the culture was performed to separate and recover
exopolysaccharides from the broth. The culture was circulated over a
0.2 μm microfiltration hollow fibre membrane with an area of 110cm2
(GE Healthcare®, CFP-2-E-3MA), until half of the culture volume
(950 mL of free-cell solution rich in EPS) was obtained as permeate.
This volume was stored at 5 °C in darkness for EPS determination. The
bioreactor was completely refilled with fresh medium immediately after
the extraction, to compensate for the loss and reach the original vo-
lume. Later, it was decided to thoroughly clean the filters daily after
use. Thus, an alkaline solution (Ultrasil® 115) was pumped through the
filters overnight and washed with demineralized water before the next
use. The milking of the reactors was performed for at least 2 weeks. A
schematic overview of the process is described in Fig. 1. For the de-
velopment and improvement of the milking process, the frequency of
the process (once per day or once every 2 days) was tested as well as the
influence of pressurization of the microfiltration cartridge (1 Bar)
during the extraction using clamps in the silicon tubes. All indoor ex-
periments were run in triplicate (n = 3).
2.3. Outdoors continuous cultivations
Outdoors cultivations were performed in 40 L closed, inclinable and
fully automatized flat panels (Green Wall Panel III, GWPIII®, photo-
synthetic surface 0.84m2, designed and built by Fotosintetica&
Microbiologica S.r.l., Italy) located in AlgaePARC, Wageningen, The
Netherlands (51°59′44.1”N 5°39′26.2″E). Cultivation was done in
176
Algal Research 34 (2018) 175–181
chemostat mode (D = 0.25d−1) during 12 h a day (from 7 am to 7 pm)
in the summer (continuously from June to August). Mass flow con-
trollers supplied air with a rate of 0.05vvm for mixing the culture. pH
was controlled by CO2 injection on demand (pH 7.2) in the airstream.
The temperature was controlled at 26 °C using a heat/cooling system
(Carrier 3ORQ-017CH). The bioreactors were facing south and were
tilted to 50° during the 12 h of the chemostat period (from 7 am to
7 pm) to capture as much light as possible. At the end of the light
period, the bioreactors were inclined to 105° to remove and avoid
biofouling formation by the action of the air bubbles. Steady state was
assumed (μ = D) after harvesting 5 times the reactor's volume (200L)
and measuring ten constant determinations of dry weight. Control and
milked reactors were running continuously for 90 days (From June to
August). The same schematic procedure for milking was followed as for
indoors experiments (Fig. 1). Once a steady state was achieved, when
the inlet of fresh medium stopped (dilution pump turned off at 7 pm),
milking of the culture was performed to separate and recover exopo-
lysaccharides from the broth. The culture was circulated over a 0.2 μm
microfiltration hollow fibre membrane with an area of 9200cm2 (GE
Healthcare®, CFP-2-E-35A). Process was stopped when half of the cul-
ture volume (20 L of free-cell solution rich in EPS) was obtained as
permeate. Permeate was stored at 5 °C in darkness for EPS determina-
tions following the same protocol as indoors experiments. The bior-
eactor was completely refilled with fresh medium immediately after the
extraction to compensate for the loss and reach the original volume.
The filters were thoroughly cleaned after use with alkaline solution
overnight (Ultrasil® 115) and demineralized water similar to the in-
doors assays. All experiments were run in duplicate (n = 2).
2.4. Analytical methods
Biomass dry weight was determined as described by Vejrazka et al.
[17]. To determine the EPS present in the culture (control reactors) and
the EPS present in the reactors before milking (and use these for de-
termining the efficiency of the extraction), a 50 mL sample of culture
was filtered manually through a 0.22 μm pore diameter filter for syr-
inges (Minisart® syringe filters Sartorius); separating the EPS fraction
from the rest of the sample. EPS contents were quantified by the Dubois
method [18]. Colony size was determined as sphere-equivalents and
measured as Volume Weighted mean (μm) using a multi-sizer (Mas-
tersizer® 2000, Malvern Instruments) as described by García-Cubero
et al. [9]. All analyses were performed in technical triplicates.
2.5. Definitions and calculations
2.5.1. Biomass productivity
Once a steady state was achieved, biomass productivity was calcu-
lated according to Eq. (1):
Pb (t ) = Cb (t ) ∗D (1)
−2 −1
where Pb(t) is biomass productivity (g m d ) at time t, Cb(t) is
biomass concentration in the reactor (g m−2) and D is the dilution rate
imposed (d−1) over 24 h.
2.5.2. EPS productivity
EPS productivity was calculated according to Eq. (2):
PEPS (t ) = CEPS (t ) ∗D (2)
−2 −1
where PEPS(t) is EPS productivity (g m d ) at time t, CEPS(t) is EPS
concentration in the reactor (g m−2) and D is the dilution rate imposed
(d−1) over 24 h.
2.5.3. PAR flux density impinging on indoor reactor's surface
The daily photon flux density of all wavelengths in PAR spectrum
impinged on reactor's surface was calculated to determine biomass and
EPS yield on light at indoors assays (Eq. (3)):
R. García-Cubero et al.
x2 2π
I0 (PAR) = ∫x1 Imax ⎛ ⎞
⎝ ∆x ⎠
Here Io(PAR) is the daily flux of PAR photons impinging in the re-
actor's surface (μmolph m−2 d−1), Imax is the max light intensity im-
pinging in the reactor surface (μmolph m−2 s−1), Δx is the number of
hours corresponding to the light period (12h) and ×1 and ×2 corre-
spond to the dawn and sunset hours of the day respectively. The daily
PAR flux density calculated was 30.25 molph m−2 d−1.
2.5.4. PAR flux density impinging on outdoor reactor's surface
The daily photon flux density of all wavelengths in PAR spectrum
impinging on outdoor reactor's surface was measured by a quantum
sensor installed on the bioreactor's surface, facing south. The experi-
mental data were similar to those compiled by the European
Photovoltaic Geographical Information System applied indoors. Thus,
the average maximum light intensity impinging on the reactor's surface
was 1100 μmolph m−2 s−1. From these data, like indoor experiments,
the daily PAR flux density measured was 30.25 molph m−2 d−1.
2.5.5. Biomass yield on light
The biomass yield on light was calculated according to Eq. (4):
Yx , ph (t ) =
Px (t )
Io (t )
where Yx,ph(t) (g molph−1) is the biomass yield on light at any time, Px
the biomass productivity (g m−2 d−1) and Io the PAR flux density im-
pinging on the reactor's surface (mol m−2 d−1).
2.5.6. EPS yield on light
EPS yield on light was determined according to Eq. (5). Here,
YEPS,ph(t) is the EPS yield on light (g molph−1), PEPS is the EPS pro-
ductivity (g m−2 d−1) and Io is the PAR flux density impinging on the
reactor's surface (mol m−2 d−1).
Algal Research 34 (2018) 175–181
Fig. 1. Schematic overview of B.braunii CCALA778 milking
process. In the figure, EPS stands for exopolysaccharide. A)
During the day, the photobioreactors were cultivated in
continuous operation (Samples EPS before milking). B) At
night, the culture was filtered continuously until half of the
bioreactor's volume was recovered and EPS was extracted.
After harvesting EPS, the volume of the bioreactor was
completed with new fresh medium (C) before sunrise of the
following day/night cycle, from which it starts with A
again.
PEPS (t )
(3)
YEPS, ph (t ) =
I0 (t ) (5)
2.6. Statistical analysis
Statistical analysis was performed using Sigma-Plot 11. One-way
ANOVA test was used to estimate differences among treatments. The
Tukey test was used post hoc to compare means. The assumptions of
normality and homoscedasticity were verified by Shapiro-Wilk and
Bartlett testes, respectively. All tests were run at a significance level of
5%.
3. Results
3.1. Indoor approach
Firstly, the effect of the milking frequency on the cultures' viability
was studied. Once the cultures achieved a steady state, they were
milked daily or once every two days for 2 weeks (Table 1). The per-
formance of the cultures was not affected by EPS extraction (p < 0.05).
The same biomass productivity was obtained in the controls, cultures
milked every day, and cultures milked every two days (23.15, 24.34,
and 23.75 g m−2 d−1, respectively). The same pattern was observed for
(4) colony size (≈130.0 μm) and EPS productivity (≈3.00 g m−2 d−1).
When the cultures were milked, it was possible to extract approximately
7% of total EPS from the broth before the extraction (0.23 g m−2 d−1
for cultures milked every day and 0.18 g m−2 d−1 for cultures milked
every two days). Directly after milking, the total EPS concentration in
the culture also was measured to close the mass balance (EPS before
milking = EPS extracted + EPS after milking). Regarding the conversion of
light into biomass (Table 2), all cultures exhibited the same efficiency,
rendering a yield of approximately 0.8 g of biomass per molph. Twelve
percent of the assimilated energy was destined to produce EPS (0.1 g
EPS per molph) and with the extraction procedure used at that time,
177
R. García-Cubero et al. Algal Research 34 (2018) 175–181

Table 1
Biomass productivity, EPS (exopolysaccharide) content before extraction, extracted EPS, EPS after extraction, and colony size in different milking treatments. The
percentage (%) is referred to the total amount of EPS produce before extraction ( ± corresponds to standard deviation, n = 3).
Biomass productivity EPS before extraction EPS extracted (%) extracted EPS after extraction (%) EPS after Colony size (μm)
(g m−2 d−1) (g m−2 d−1) (g m−2 d−1) (g m−2 d−1) extraction

Control 23.15 ± 2.37 2.85 ± 0.9 – – – – 134.2 ± 2.9

Milking every day 24.34 ± 0.6 3.21 ± 0.6 0.23 ± 0.08 7.16 2.79 ± 0.6 92.84 129.8 ± 3.6
Milking every two days 23.75 ± 0.6 2.61 ± 0.6 0.18 ± 0.08 6.89 2.31 ± 0.6 93.11 130.6 ± 1.4
Milking and cleaning 23.27 ± 0.4 3.02 ± 0.2 0.36 ± 0.06 11.90 2.55 ± 0.3 88.10 92.1 ± 5.4
every day
Milking, cleaning and 12.47 ± 0.5 0.53 ± 0.1 0.23 ± 0.06 43.39 0.28 ± 0.06 56.61 50.6 ± 2.1
pressure (1Ba) every
day

barely 0.8–0.9% of the assimilated energy was recovered as EPS (7.8
and 5.9 mg EPS extracted per molph for milking daily and every two days,
respectively). From the data obtained, the frequency of extraction did
not affect the culture's viability. Thus, a daily milking was selected for
the next step in the optimization of extraction. The extraction effi-
ciency, however, required further be improvement. One of the possible
reasons for such a low efficiency could be the clogging of pores by the
biomass during the milking process. For this reason, we focused on how
to increase the efficiency by cleaning the filters after use with an al-
kaline solution.
When both the milking and the subsequent cleaning of the micro-
filtration membranes were done daily, the biomass and EPS pro-
ductivity were the same as the controls, yielding respectively 23.27 and
3.02 g m−2 d−1 (Table 1). However, with this new approach, the ex-
traction of EPS increased from 7% to 11.9% (from 0.23 g m−2 d−1 to
0.36 g m−2 d−1). The average size of the colony decreased to 92 μm
(30% decrease compared to the colony size in the control reactor)
(Table 1). In this new approach, the efficiency in converting light into
biomass and EPS remained the same but it was possible to recover al-
most two times the light assimilated as EPS than previous approaches
(10.82 mg EPS extracted molph−1) (Table 2).
A third step to increase the efficiency in EPS recovery was tested by
applying pressure in the microfiltration system during the milking
(Table 1)., Approximately 45% of the total EPS present in the broth was
recovered (0.23 g EPS extracted m−2 d−1 of 0.53 g EPS broth m−2 d−1).
Although the extraction efficiency increased, the biomass and EPS
productivities were reduced 50 and 80% when compared to controls
(Controls: 23.15 g biomass m−2 d−1 vs Filtration with pressure: 12.47 g
biomass m−2 d−1; Controls 2.85 g EPS m−2 s−1 vs Filtration with
pressure: 0.53 g m−2 d−1, respectively). The same trend was observed
for the yields on light of biomass and EPS (Table 2). Thus, only 0.37 g of
biomass was generated per mol of PAR photons assimilated and barely
3% was destined to EPS production (0.01 g EPS molph−1). In this fra-
mework, the recovery of the energy assimilated via photosynthesis was
barely of 1.8% (6.92 mg EPS extracted m−2 d−1).
Given the results above, the extraction outdoors was done daily with
no additional pressure and was followed by flushing of the filters with
alkaline solution overnight.
3.2. Outdoor experiments
Based on the results obtained indoors, a continuous extraction of
EPS was done in outdoors continuous cultures of B. braunii CCALA778
during the summer of 2016 in AlgaePARC (Wageningen, NL). For both
control and milked cultures, biomass productivity was similar, ren-
dering 17 g m−2 d−1 (Table 3). Regarding EPS productivity, the same
trend was observed for both conditions, yielding a productivity of 4 g
EPS m−2 d−1. However, we could extract barely 3% of the total EPS
content (0.12 g EPS m−2 d−1). The size of the colonies was not affected
by extraction and cultures showed the same size in both conditions (in
the range 120–130 μm). Outdoors, one mol of PAR photons was needed
to produce approximately 0.58 g of biomass (Table 4). Thus, 20% of this
energy was used for EPS production (0.12 g EPS molph−1), while only
0.7% of the assimilated energy was recovered in the EPS by milking
(4 mg EPS m−2 d−1).
4. Discussion
Some strains of B. braunii have been studied regarding their suit-
ability for production and milking of hydrocarbons [14]. Milking of
hydrocarbons is generally performed by organic solvent extraction or
supercritical CO2 [19, 20]. Similarly, we studied a process for in situ
extracting exopolysaccharides produced by B. braunii. The choice of
B.braunii CCALA778 as a source of exopolysaccharides was done based
on promising results obtained by Gouveia et al. and García-Cubero et al.
[8, 9]. The EPS fraction of B. braunii CCALA 778 is rich in galactose and
fucose and contains also glucuronic and galacturonic acids [8, 9]. The
commercial value of the sugars from B. braunii will come from assessing
the whole complex matrix of these exopolysaccharides and their spe-
cific characteristics to be used as surfactants and emulsifiers [21]. Ad-
ditionally, these sugars could be used in cosmetics, healthcare and
specialty markets, such as food ingredients. Other microalgae and cy-
anobacteria species, such as Anabaena, Porphyridium or Nitzschia, have
also been proposed for EPS production [22, 23], but milking has never
been reported with these species.
Extracting EPS present a natural advantage of not needing organic
solvents. The separation, thus, might be based on centrifugation or

Table 2
Biomass, EPS (exopolysaccharide) before extraction and EPS (exopolysaccharide) extracted yield on light of the different treatments for milking indoors. The
percentage (%) is referred to the amount of energy destined to produce exopolysaccharide from the energy used to produce biomass. ( ± corresponds to standard
deviation, n = 3).
Biomass yield on light (%) EPS yield on light before extraction (%) EPS extracted yield on light (%)
(g molph−1) (g molph−1) (mg molph−1)

Control 0.76 ± 0.08 – 0.09 ± 0.02 – – –

Milking every day 0.80 ± 0.02 100 0.10 ± 0.02 12.50 7.14 ± 2.4 0.89
Milking every two days 0.78 ± 0.02 100 0.09 ± 0.01 11.53 5.41 ± 2.4 0.69
Milking and cleaning every day 0.70 ± 0.01 100 0.09 ± 0.0 12.85 10.82 ± 1.8 1.54
Milking, cleaning and pressure (1Ba) 0.37 ± 0.01 100 0.02 ± 0.0 0.05 6.92 ± 1.8 1.87
every day

178
R. García-Cubero et al. Algal Research 34 (2018) 175–181

Table 3
Biomass productivity, EPS (exopolysaccharide) content before extraction, extracted and after extraction and colony size outdoors. The percentage (%) is referred to
the total amount of EPS (exopolysaccharide) produce before extraction. ( ± corresponds to standard deviation, n = 3).
Biomass productivity EPS before extraction EPS extracted (%) EPS after extraction (%) Colony size (μm)
(g m−2 d−1) (g m−2 d−1) (g m−2 d−1) (g m−2 d−1)

Control 17.5 ± 1.8 3.7 ± 1.8 – – – – 129.8 ± 4.1

Milking and cleaning 17.3 ± 2.6 4.1 ± 1.8 0.12 ± 0.06 2.92 4.0 ± 0.9 97.56 121.3 ± 6.4
every day

Table 4
Biomass, EPS (exopolysaccharide) before extraction and EPS (exopolysaccharide) extracted yield on light outdoors. The percentage (%) is referred to the amount of
energy destined to produce exopolysaccharide from the energy used to produce biomass. ( ± corresponds to standard deviation, n = 2).
Biomass yield on (%) EPS yield on light before (%) EPS extracted yield on (%)
light (g molph−1) extraction (g molph−1) light (mg molph−1)

Control 0.58 ± 0.06 – 0.12 ± 0.06 – – –

Milking and cleaning 0.57 ± 0.08 100 0.13 ± 0.06 22.80 4±1 0.7
every day

filtration in order to milk these components. Therefore, the main scope
of our research was to develop and improve a technology for EPS
milking without compromising the cell's viability. Initially, centrifuga-
tion trials were tested to separate EPS from biomass. However, cen-
trifugation was discharged because of its relatively high energy con-
sumption and therefore being not economically sustainable in an
industrial framework [24]. This preliminary result led to choose for
microfiltration as an alternative in the extraction process. Thus, a mi-
crofiltration cartridge system with a pore diameter of 0.2 μm was se-
lected as it could be used to separate efficiently the EPS from the broth
[25]. This approach was intended to reduce the cost of EPS downstream
processes because microfiltration is known to be a low energy de-
manding process in comparison to centrifugation [26], as well as to
maintain the cultures' viability.
B. braunii grows slowly while simultaneously producing and ex-
creting EPS [14]. Thus, a continuous cultivation and milking could be a
suitable option to reduce EPS harvesting intervals and, consequently, to
achieve high EPS productivities. It has been reported that batch op-
eration might increase the EPS content in B.braunii CCALA778 cultures
owing to N starvation (and therefore the unbalance of the ratio C/N,
leading to C-rich store compounds as lipids or carbohydrates), which
typically occurs at the end of the lag phase or during stationary phase
[27, 28]. However, our results showed that the application of con-
tinuous cultivation increased EPS productivity in B.braunii CCALA778
up to 0.13 g L−1 d−1 (after conversion from areal productivity to vo-
lumetric productivity). This is up to 10 times higher than results ob-
tained in batch with the same strain by Gouveia et al. (a content of
0.315 g total carbohydrates after 30 days of cultivation, meaning a
volumetric EPS productivity of 0.01 g L−1 d−1) [8]. It's important to
notice that Gouveia et al. focused on total carbohydrates and not on
exopolysaccharides, most likely the EPS productivity in their results
was lower. Our results showed that it is possible to achieve a pro-
ductivity of 0.08 g EPS L−1 d−1 in batch cultivations (from the batch
phase of 15 days before shifting to continuous cultures). In addition, we
can estimate the same maximum extraction efficiency as in continuous
cultivation (43%) at the end of the lag phase, resulting in an EPS pro-
ductivity of 0.0344 g EPS g L−1 d−1. Nevertheless, this approach would
lead to a lower yield in comparison to an extraction of continuous
cultures, as our results showed. Our data agreed with those from Klei-
negris et al. [29], which demonstrated that continuous extraction of a
product can induce product formation. This positive effect might be
induced by alleviating feed-back inhibition or decreased product de-
gradation.
Considering the microalgal cells as photosynthetic cell factories
[30], it is interesting to determine the EPS yield on light and compare it
to the biomass yield on light. These data help to understand the carbon
partitioning in the cell, the achieved photosynthetic efficiencies, and
the energy returned on energy invested (EROI). These parameters can
indicate if EPS milking from B.braunii is economically feasible [31] and
determine a more accurate life cycle analysis (LCA) [32]. The EPS yield
on light obtained in this work corroborates previously reports for
B.braunii CCALA 778: the EPS yield on light increases with increasing
light intensity [9]. At low light intensities (800μmolPAR m−2 s−1), the
percentage of energy destined to produce EPS varies from 6 to 8%of the
energy used to produce biomass. At high light intensities (2000μmolPAR
m−2 s−1), this percentage increases to > 20%. Our results showed that
the percentage of energy destined to produce EPS from the energy used
to produce biomass is close to 12% at 1100μmolPAR m−2 s−1, when the
milking process did not affect the cell's viability (no pressure applied in
the extraction process). However, this percentage of conversion in-
creased outdoors to > 20%. This might be caused by the presence of
grazers typically found in outdoor cultivations, which could trigger an
increase in EPS production as a protection agent [33–35]. Regarding
the efficiency in converting light to biomass, B.braunii CCALA778
showed higher values in comparison to Chloroccocum littorale (0.45 g
molph−1) [36] and in the same range of Acutodesmus obliquus [37].
These data show that B. braunii is a microalga with a high light as-
similation. However, B. braunii is characterized by its slow growth,
therefore, the energy assimilated by this microalga is not driven to
growth but to produce other compounds such as hydrocarbons or EPS
[2].
Similar to the microalgae Scenedesmus vacuolatus and Chlorella vul-
garis [38, 39], B.braunii CCALA778 was confirmed to thrive in con-
tinuous operation [9]. In our previous work, B.braunii CCALA778 ren-
dered up to 2.13 g EPS m−2 d−1 (volumetric productivity of 0.09 g EPS
L−1 d−1) when it was cultivated at low light intensities (800μmolph
m−2 s−1) and 6.88 g EPS m−2 d−1 (volumetric productivity of 0.29 g
EPS L−1 d−1) at high light intensities (2000μmolph m−2 s−1). The light
intensity applied during indoor experiments in the present work was
1100μmolph m−2 s−1, corresponding to the average light regime mea-
sured in AlgaePARC during the months of June/July/August of the last
ten years (From 2006 to 2016). Clearly, the EPS productivity achieved
in the current work (3 g EPS m−2 d−1) at medium light intensity
(1100μmolph m−2 s−1) revealed the linear relationship between light
intensity and EPS production in B.braunii CCALA778 continuous cul-
tures when temperature is controlled at 26 °C.
Literature does not provide too many examples of outdoor cultures
of B.braunii strains and the ones reported are mainly under batch op-
eration [40, 41]. Interestingly, Bazaes et al. demonstrated the feasibility
of outdoor continuous cultures of B.braunii in the Atacama desert

179
R. García-Cubero et al.
(Chile) [42]. However, biomass productivity was poor and barely
achieved 0.02 g biomass L−1 d−1, 18 times lower than our results
(0.36 g biomass L−1 d−1). The low productivities achieved by Bazaes
et al. might be explained by the lack of temperature and pH control in
their outdoor reactors.
Although our results regarding outdoor exopolysaccharide pro-
ductivity were promising, its recovery was poor and the efficiency in
the extraction dropped 3 times compared to the indoor results (from
12% EPS recovery indoors to barely 3% outdoors). The loss in efficiency
of the microfiltration cartridge outdoors can be due to different factors,
such as EPS/biomass ratio (indoors was lower than outdoors), the size
of the colonies (smaller indoors than outdoors) or increase of algogenic
organic matter (AOM) and algal debris [24, 43]. Different strategies to
avoid a reduction in the filtration efficiency have been extensively
studied [44]. The proposed methods include a pre-treatment of the
feedwater (microalgal broth) by using pH, Ca2+, coagulants, oxidants,
and adsorbents, and improving the hydrodynamic conditions as feed-
flux or aeration [24]. One of the major responsible agents in membrane
fouling is the algogenic organic matter (AOM) that naturally occurs in
microalgal cultures. This released organic matter is produced by normal
growth or in response to a changing environment [45], but the natural
trend is that AOM content increases alongside cultivation aging [46]. In
our case, outdoor reactors were running for three months (June to
August), compared to 4–5 weeks for the indoor experiments. Under-
standably, AOM increased in the broth after such a long period, making
the filtration more difficult. This could explain the differences in EPS
extraction yields between indoor and outdoor experiments. Moreover,
it has recently been demonstrated that the size of the colonies influ-
ences the hydrocarbons extractability in hydrocarbon-producing strains
of B. braunii [43, 47]. Our results indicated that the size of the colonies
was bigger outdoors than indoors in the milked cultures (increasing
from 92 μm indoors to 121 μm outdoors); although the size of colonies
in both controls barely shifted (134μmindoors; 129.8 μm, outdoors).
Therefore, our suggestion is that AOM influences the size of the colonies
by making the cells more attached to each other and therefore, in-
creasing the size of the colonies. Bigger colonies provoke more fouling
in the filters and consequently, a reduction in the EPS extractability.
The hydrodynamic shear force resulting from applying a pressure in the
extraction filter affects the union strength between colonies. Therefore,
the colony size reduces, the milking yield increases, but the cell's via-
bility is also affected [19].
Still, there is a big gap to overcome the technical difficulties in
microalgal milking and specifically in EPS extraction from B. braunii.
Recently, several studies have been published regarding how the nature
of the microalgae species and EPS composition can affect the efficiency
in the filtration [48, 49] as well as novel approaches for increasing the
efficiency in the recovery as ultrasound-assisted extraction (UAE) and
microwave-assisted extraction (MAE) [50]. More work should be done
to increase the EPS extraction efficiency in B. braunii CCALA778 out-
door cultures in order to make this a viable process.
5. Conclusions
In this paper, we demonstrated that it is possible to cultivate
B.braunii CCLA778 in continuous operation, achieving a steady state,
both indoors and outdoors. In addition, a continuous EPS extraction
was applied without compromising cell viability. Our results indicate
that potentially 12% of EPS produced by this microalga can be milked.
The application of pressure increased the EPS extractability but da-
maged the cells, thus reducing cell viability. Despite these promising
findings, more work must be accomplished in the future to increase
outdoor production yield and extraction efficiency.
Acknowledgements
This work has been supported financially by the European
180
Algal Research 34 (2018) 175–181
Community under the Seventh framework programme (Project
SPLASH: http://www. eu-splash.eu; contract number 311956).
Declaration of authors contributions
García-Cubero: Conception, experimental design and set-up, ex-
perimental work, analysis and interpretation of the data, statistical
analysis of the data, drafting of the manuscript and final approval of the
manuscript. Weiliang Wang, Judit Martín and Elisabeth Bermejo: ex-
perimental work and final approval of the manuscript. Lolke Sijtsma:
Obtaining of funding, analysis and interpretation of the data and final
approval of the manuscript. Arnoud Togtema: experimental work,
conception, experimental design, analysis and interpretation of the data
and final approval of the manuscript. María J. Barbosa: Obtaining of
funding, conception, experimental design, analysis and interpretation
of the data, critical revision of the manuscript and final approval of the
manuscript. Dorinde M.M. Kleinegris: Conception, experimental design,
analysis and interpretation of the data, critical revision of the manu-
script and final approval of the manuscript.
Conflict of interest statement
Authors declare no conflict of interest.
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights applicable.
Declaration of authors agreement to authorship and submission of
the manuscript
Authors agree to authorship and submission of the manuscript.
References
[1] P. Metzger, C. Largeau, Botryococcus braunii: a rich source for hydrocarbons and
related ether lipids, Appl. Microbiol. Biotechnol. 66 (2005) 486–496.
[2] A. Banerjee, R. Sharma, M.Y. Chisti, U.C. Banerjee, Botryococcus braunii: a re-
newable source of hydrocarbons and other chemicals, Crit. Rev. Biotechnol. 22
(2002) 245–279.
[3] Q. Hu, N. Kurano, M. Kawachi, I. Iwasaki, S. Miyachi, Ultrahigh-cell-density culture
of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor, Appl.
Microbiol. Biotechnol. 49 (1998) 655–662.
[4] M.M. Watanabe, Y. Tanabe, Biology and industrial potential of Botryococcus braunii,
Handbook of Microalgal Culture: Applied Phycology and Biotechnology, 2013, pp.
369–387.
[5] S. Chaudry, P.A. Bahri, N.R. Moheimani, Selection of an Energetically More Feasible
Route for Hydrocarbon Extraction from Microalgae – Milking of B. braunii as a Case
Study, Computer Aided Chemical Engineering, (2016), pp. 1545–1550.
[6] Y. Ge, J. Liu, G. Tian, Growth characteristics of Botryococcus braunii 765 under high
CO2 concentration in photobioreactor, Bioresour. Technol. 102 (2011) 130–134.
[7] T. Manchanda, R. Tyagi, D.K. Sharma, Application of nutrient stress conditions for
hydrocarbon and oil production by Botryococcus braunii, Biofuels (2016) 1–10.
[8] J.D. Gouveia, J. Ruiz, L.A.M. van den Broek, T. Hesselink, S. Peters,
D.M.M. Kleinegris, A.G. Smith, D. van der Veen, M.J. Barbosa, R.H. Wijffels,
Botryococcus braunii strains compared for biomass productivity, hydrocarbon and
carbohydrate content, J. Biotechnol. 248 (2017) 77–86.
[9] R. García-Cubero, I.T.D. Cabanelas, L. Sijtsma, D.M.M. Kleinegris, M.J. Barbosa,
Production of exopolysaccharide by Botryococcus braunii CCALA 778 under la-
boratory simulated Mediterranean climate conditions, Algal Res. 29 (2018)
330–336.
[10] J.A. Galbis, M.D.G. García-Martín, M.V. De Paz, E. Galbis, Synthetic Polymers from
Sugar-based Monomers, Chem. Rev. 116 (2016) 1600–1636.
[11] S. Chaudry, P.A. Bahri, N.R. Moheimani, Techno-economic analysis of milking of
Botryococcus braunii for renewable hydrocarbon production, Algal Res. 31 (2018)
194–203.
[12] C. Griehl, C. Kleinert, C. Griehl, S. Bieler, Design of a continuous milking bioreactor
for non-destructive hydrocarbon extraction from Botryococcus braunii, J. Appl.
Phycol. 27 (2015) 1833–1843.
[13] M. Ota, Y. Hiraga, Y. Hamano, F. Hishinuma, I. Ushiki, T. Ono, Y. Sato, H. Inomata,
Continuous wet-extraction of hydrocarbon from botryococcus Braunii, Kagaku
Kogaku Ronbunshu 44 (2018) 103–106.
[14] E. Yildiz-Ozturk, O. Yesil-Celiktas, Supercritical CO2 extraction of hydrocarbons
from Botryococcus braunii as a promising bioresource, J. Supercrit. Fluids 130
(2017) 261–266.
R. García-Cubero et al.
[15] S. Tsutsumi, Y. Saito, Y. Matsushita, H. Aoki, Effect of Mechanical Pretreatment on
Hydrocarbon Extraction from Concentrated Wet Hydrocarbon-Rich Microalga,
Botryococcus braunii, Energy Fuel 32 (2018) 1761–1770.
[16] G. Benvenuti, R. Bosma, F. Ji, P. Lamers, M.J. Barbosa, R.H. Wijffels, Batch and
semi-continuous microalgal TAG production in lab-scale and outdoor photo-
bioreactors, J. Appl. Phycol. 28 (2016) 3167–3177.
[17] C. Vejrazka, M. Janssen, M. Streefland, R.H. Wijffels, Photosynthetic efficiency of
Chlamydomonas reinhardtii in flashing light, Biotechnol. Bioeng. 108 (2011)
2905–2913.
[18] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method
for determination of sugars and related substances, Anal. Chem. 28 (1956)
350–356.
[19] B.A. Jackson, P.A. Bahri, N.R. Moheimani, Repetitive non-destructive milking of
hydrocarbons from Botryococcus braunii, Renew. Sust. Energ. Rev. 79 (2017)
1229–1240.
[20] M.B. Tasić, L.F.R. Pinto, B.C. Klein, V.B. Veljković, R.M. Filho, Botryococcus braunii
for biodiesel production, Renew. Sust. Energ. Rev. 64 (2016) 260–270.
[21] J.J. Paniagua-Michel, J. Olmos-Soto, E.R. Morales-Guerrero, Algal and microbial
exopolysaccharides: New insights as biosurfactants and bioemulsifiers, Adv. Food
Nutr. Res. (2014) 221–257.
[22] J. Moreno, M.A. Vargas, H. Olivares, J. Rivas, M.G. Guerrero, Exopolysaccharide
production by the cyanobacterium Anabaena sp. ATCC 33047 in batch and con-
tinuous culture, J. Biotechnol. 60 (1998) 175–182.
[23] A. Penna, S. Berluti, N. Penna, M. Magnani, Influence of nutrient ratios on the in
vitro extracellular polysaccharide production by marine diatoms from the Adriatic
Sea, J. Plankton Res. 21 (1999) 1681–1690.
[24] Y. Zhang, Q. Fu, Algal fouling of microfiltration and ultrafiltration membranes and
control strategies: a review, Sep. Purif. Technol. 203 (2018) 193–208.
[25] M.R. Bilad, D. Vandamme, I. Foubert, K. Muylaert, I.F.J. Vankelecom, Harvesting
microalgal biomass using submerged microfiltration membranes, Bioresour.
Technol. 111 (2012) 343–352.
[26] M.R. Bilad, H.A. Arafat, I.F.J. Vankelecom, Membrane technology in microalgae
cultivation and harvesting: a review, Biotechnol. Adv. 32 (2014) 1283–1300.
[27] I. Gifuni, G. Olivieri, A. Pollio, A. Marzocchella, Identification of an industrial mi-
croalgal strain for starch production in biorefinery context: the effect of nitrogen
and carbon concentration on starch accumulation, New Biotechnol. 41 (2018)
46–54.
[28] L.M. Schüler, P.S.C. Schulze, H. Pereira, L. Barreira, R. León, J. Varela, Trends and
strategies to enhance triacylglycerols and high-value compounds in microalgae,
Algal Res. 25 (2017) 263–273.
[29] D.M.M. Kleinegris, M. Janssen, W.A. Brandenburg, R.H. Wijffels, Two-phase sys-
tems: potential for in situ extraction of microalgal products, Biotechnol. Adv. 29
(2011) 502–507.
[30] R.H. Wijffels, M.J. Barbosa, An outlook on microalgal biofuels, Science 329 (2010)
796–799.
[31] L. Barsanti, P. Gualtieri, Is exploitation of microalgae economically and en-
ergetically sustainable? Algal Res. 31 (2018) 107–115.
[32] F. Ketzer, J. Skarka, C. Rösch, Critical Review of Microalgae LCA Studies for
Bioenergy Production, Bioenergy Research 11 (2018) 95–105.
[33] Y. Wang, Y. Gong, L. Dai, M. Sommerfeld, C. Zhang, Q. Hu, Identification of harmful
protozoa in outdoor cultivation of Chlorella and the use of ultrasonication to control
181
Algal Research 34 (2018) 175–181
contamination, Algal Res. 31 (2018) 298–310.
[34] V. Montemezzani, I.C. Duggan, I.D. Hogg, R.J. Craggs, A review of potential
methods for zooplankton control in wastewater treatment High Rate Algal Ponds
and algal production raceways, Algal Res. 11 (2015) 211–226.
[35] S.E.A. Zerrifi, F.E. Khalloufi, B. Oudra, V. Vasconcelos, Seaweed bioactive com-
pounds against pathogens and microalgae: potential uses on pharmacology and
harmful algae bloom control, Mar. Drugs 16 (2018).
[36] I.T.D. Cabanelas, P.M. Slegers, H. Böpple, D.M.M. Kleinegris, R.H. Wijffels,
M.J. Barbosa, Outdoor performance of Chlorococcum littorale at different locations,
Algal Res. 27 (2017) 55–64.
[37] G.M. León-Saiki, I.M. Remmers, D.E. Martens, P.P. Lamers, R.H. Wijffels, D. van der
Veen, The role of starch as transient energy buffer in synchronized microalgal
growth in Acutodesmus obliquus, Algal Res. 25 (2017) 160–167.
[38] R. García-Cubero, J. Moreno-Fernández, M. García-González, Modelling growth and
CO2 fixation by Scenedesmus vacuolatus in continuous culture, Algal Res. 24, Part
A (2017) 333–339.
[39] R. García-Cubero, J. Moreno-Fernández, M. García-González, Potential of Chlorella
vulgaris to Abate Flue Gas, Waste Biomass Valoriz. (2017) 1–5.
[40] V. Ashokkumar, R. Rengasamy, Mass culture of Botryococcus braunii Kutz. Under
open raceway pond for biofuel production, Bioresour. Technol. 104 (2012)
394–399.
[41] A. Ranga Rao, G.A. Ravishankar, R. Sarada, Cultivation of green alga Botryococcus
braunii in raceway, circular ponds under outdoor conditions and its growth, hy-
drocarbon production, Bioresour. Technol. 123 (2012) 528–533.
[42] J. Bazaes, C. Sepulveda, F.G. Acién, J. Morales, L. Gonzales, M. Rivas, C. Riquelme,
Outdoor pilot-scale production of Botryococcus braunii in panel reactors, J. Appl.
Phycol. 24 (2012) 1353–1360.
[43] S. Tsutsumi, Y. Saito, Y. Matsushita, H. Aoki, Investigation of colony disruption for
hydrocarbon extraction from Botryococcus braunii, Fuel Process. Technol. 172
(2018) 36–48.
[44] J.B. Castaing, A. Massé, M. Pontié, V. Séchet, J. Haure, P. Jaouen, Investigating
submerged ultrafiltration (UF) and microfiltration (MF) membranes for seawater
pre-treatment dedicated to total removal of undesirable micro-algae, Desalination
253 (2010) 71–77.
[45] G.G. Leppard, The characterization of algal and microbial mucilages and their ag-
gregates in aquatic ecosystems, Sci. Total Environ. 165 (1995) 103–131.
[46] R.K. Henderson, A. Baker, S.A. Parsons, B. Jefferson, Characterisation of algogenic
organic matter extracted from cyanobacteria, green algae and diatoms, Water Res.
42 (2008) 3435–3445.
[47] S. Tsutsumi, M. Yokomizo, Y. Saito, Y. Matsushita, H. Aoki, Mechanical cell dis-
ruption of microalgae for investigating the effects of degree of disruption on hy-
drocarbon extraction, Asia Pac. J. Chem. Eng. 12 (2017) 454–467.
[48] M. Larronde-Larretche, X. Jin, Microalgal biomass dewatering using forward os-
mosis membrane: Influence of microalgae species and carbohydrates composition,
Algal Res. 23 (2017) 12–19.
[49] M. Shekhar, A. Shriwastav, P. Bose, S. Hameed, Microfiltration of algae: Impact of
algal species, backwashing mode and duration of filtration cycle, Algal Res. 23
(2017) 104–112.
[50] C. Delattre, G. Pierre, C. Laroche, P. Michaud, Production, extraction and char-
acterization of microalgal and cyanobacterial exopolysaccharides, Biotechnol. Adv.
34 (2016) 1159–1179.