Bioresource Technology 129 (2013) 430438

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Medium recycling for Nannochloropsis gaditana cultures for aquaculture

C.V. Gonzlez-Lpez a, M.C. Cern-Garca a,, J.M. Fernndez-Sevilla a, A.M. Gonzlez-Cspedes b,
J. Camacho-Rodrguez a, E. Molina-Grima a
a
b

Department of Chemical Engineering, University of Almera, E04120 Almera, Spain

Department of Greenhouse Technology, Estacin Experimental Fundacin Cajamar, Almera E04710, Spain

h i g h l i g h t s
" A methodology to recirculate medium in microalgae cultures is developed.
" The most adequate sterilization method is established.
" The recirculation reduces the demand of nutrients cutting costs in the process.
" N. gaditana continuous cultures were maintained using the recirculated medium.
" The biomass biochemical composition resulted of high interest for aquaculture.

a r t i c l e

i n f o

Article history:
Received 1 August 2012
Received in revised form 12 November 2012
Accepted 16 November 2012
Available online 28 November 2012
Keywords:
Medium recycling
Sterilization
Microalgae
Aquaculture

a b s t r a c t
Nannochloropsis gaditana is a good producer of proteins and valuable fatty acids for aquaculture. Recycling of culture medium is interesting for microalgae commercial production as it cuts costs and prevents
environmental contamination. The recycled medium must be sterilized to prevent the buildup of
unwanted metabolites and microorganisms. We tested several sterilization methods: ltration, ozonation, chlorination, addition of hydrogen peroxide and heating. Results showed that the most successful
method is ozonation lowering the bacterial load to 1.9 103 CFUs/mL, which is 1000-fold and 10-fold lower
than the supernatant obtained after harvesting and the initial ltered medium, respectively. Continuous
cultures of N. gaditana were grown using this recirculated supernatant. A maximum biomass productivity
of 0.8 g/L/d composed of 50% proteins and 40% lipids with more than 3% d.w. EPA was obtained making
this biomass very interesting for aquaculture.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
The production of microalgae is essential for the commercial
cultivation of larvae of mollusks, crustaceans and sh. Microalgae
can be used directly as live food (for mollusks and crustaceans)
or indirectly as food for zooplankton species, such as the rotifer
Brachiomus plicatilis and nauplii of Artemia, used as prey for sh
larvae (Benemann, 1992). The microalgae used as food in the early
larval stages of sh should ensure the nutritional requirements of
the species, particularly in essential compounds like amino acids or
polyunsaturated fatty acids. The microalgal diet must contain high
levels of polyunsaturated fatty acids as docosahexanoic acid (DHA
22:6n3), eicosapentanoicacid (EPA 20:5n3) and araquidonic acid
(AA 20:4n6) to ensure a good larvae growth and high levels of survival (Navarro and Villanueva, 2003; Morais et al., 2005).

Corresponding author. Tel.: +34 950 015981; fax: +34 950 015484.
E-mail address: mcceron@ual.es (M.C. Cern-Garca).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.11.061

The maintenance of axenic mass cultures of microalgae requires

the sterilization of large volumes of seawater and thus of a method
that can be implemented in the large scale. Kawachi and Nel
(2005) reviewed several techniques to sterilize seawater. Among
the methods discussed by these authors are ltration, application
of ultraviolet radiation (UV), addition of sodium hypochlorite and
pasteurization. Other techniques such as membrane-based separation methods have been suggested (Rathore and Shirke, 2011) but
these are more difcult to implement in large-scale culture systems and especially in seawater.
Many aquaculture facilities use ltration, UV irradiation or a
combination of both methods to sterilize seawater. Ozonation
and ultraviolet irradiation (UV) are the most frequently used methods for viral control in land based aquacultural systems. These two
methods can be used to eliminate pathogens in the inlet seawater
culture medium, in the supernatant and in the recirculated water
(the supernatant obtained after harvesting). Disinfection by ozonation and UV irradiation are also used in other aquacultural applications, e. g., to reduce or eliminate the presence of potential

C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

pathogens associated with live prey, such as rotifers, in marine larval production systems or to disinfect the surface of sh eggs
(Grotmol and Totland, 2000). The ltration only eliminates particles of the size of the smallest lter and does not eliminate viruses.
A chemical sterilization method is the chlorination of water with
bleach (sodium hypochlorite) for several hours, followed by the removal of the excess of chlorine by the addition of sodium thiosulphate and aeration (Kawachi and Nel, 2005). Regarding thermal
disinfection, pasteurization has proved to be an effective method
for two species of algae: Rhodomonas lens (CCMP 739) and R. salina
(CCMP 1319) (Rhodes et al., 2008).
The use of ozone began in the 1990s, with the purication of
drinking water and ever since it has become widespread. Ozone
is used for disinfection of bottled water and in the oxidative
decomposition of harmful impurities in industrial and domestic
sewage (Martnez et al., 2011). It is a strong disinfectant with a
high oxidation potential and it is one of the most effective and frequently used for deactivating pathogens in drinking water treatments (Langlais et al., 1991). Ozone also acts against various
cellular substances including lipids, peptidoglycans, enzymes and
viruses. It is a powerful disinfectant against bacteria, fungi, etc.
and it can therefore be used to satisfy the highest disinfection standards (Tyrrell et al., 1995).
The production of microalgae at large-scale with medium recycling has not yet been commercialized. The life-cycle impacts of
large-scale microalgae productions are being debated, specially
the impact on water usage, i. e., the water consumption per hectare of land used for algal feedstock production. As far as we know,
the life-cycle of water when using seawater and recycling the
harvested water to produce microalgae in aquaculture has not
been established yet. The nearer attempts are those of Yang
et al. (2011) and Jung and Lovitt (2010) who recycled the harvested water for microalgae production with the objective to obtain biodiesel and PUFAs, respectively. Therefore, if the
supernatant obtained from the harvest of the biomass is not recirculated to the system, high amounts of nutrients are thrown out.
It is possible to reuse those nutrients by using closed systems to
recirculate the water and take the most of the nutrients not
consumed.
The biggest challenge of this issue is to develop a method to
sterilize the supernatant obtained from the harvested biomass of
microalgae cultures and to recirculate it to the system as to work
in a closed circuit. The harvesting process involves the presence
of suspended solids in the supernatant. These solids are organic
matter that accumulates over time. So it is necessary to study
the inuence of this organic matter on the microalga growth. For
example, Chlorella sp. uses organic matter to promote its growth
and the subsequent reduction of chemical oxygen demand (COD)
(Li et al. 2011). COD is an important and easily reproducible
parameter in the analysis of domestic and industrial wastewaters
in terms of the evaluation of their pollution levels. Nevertheless,
the methodology for its determination is not well adapted to seawater so the results are not reliable. Saral and Goncalog (2008)
developed a method to determine COD of saline wastewater (up
to 14 gNaCl/L) but it involved the use of toxic chemicals. On the
other hand, Aziz and Tebbut (1980) assessed that the total organic
carbon (TOC) is an alternative to COD measurements. The TOC
measurement is not dependent on the salt content of the sample.
So to account the organic matter, the TOC content would be used
as the preferable parameter.
The aim of this work is to develop a methodology at laboratory
scale that allows recirculating the supernatant obtained after harvesting microalgae cultures. For that, it is necessary to establish the
best sterilization method to be used in order to avoid any inuence
on the microalgae production and/or on their biochemical composition. The experimentation has been designed bearing in mind

431

that the technique selected will be later implemented in a large

closed photobioreactor of commercial scale. In this way, the supernatant could be used as a nutrient source for the production of high
value biomass reducing the demand of nutrients and emitting cleaner efuents. Once the optimal technique for recirculation is established, it will be possible in future contribution to study long-term
recycling and its economic impact in large-scale systems.
2. Methods
2.1. Sterilization methods
The articial seawater culture medium used was provided by
the microalgal production facility Estacin Experimental Fundacin Cajamar property of Cajamar (Paraje Las Palmerillas, Almera,
Spain). The composition of this medium is the same as the commercial Algal medium (Bionova, Santiago de Compostela, Spain)
prepared at 8 mM KNO3 concentration but using agricultural fertilizers instead of pure grade chemicals. The tests were performed at
laboratory scale in 100 mL Erlenmeyer asks containing 50 mL of
the seawater culture medium. They were incubated on an orbital
shaker (Orbital midi, Ovan, Barcelona, Spain) at 100 rpm without
aeration under 25 C and aseptic conditions during 7 days.
Six different techniques were used to sterilize the medium:
heating and addition of four different chemicals. The seawater culture medium was rst sterilized in autoclave (Presoclave-II, J.P.
Selecta, Barcelona, Spain) at 126 C for 20 min in order to guarantee the sterilization used as the control test. The chemical sterilization was performed by the addition of four compounds: sodium
hypochlorite (PANREAC Qumica S.L.U., Barcelona, Spain), dichloroisocyanurate (Suavinex C.N. 178665, Alicante, Spain), ozone
(Ozone generator 3060, ETRON Ecology S.L., Spain) and hydrogen
peroxide (PANREAC Qumica S.L.U., Barcelona, Spain). The chlorination was carried out with different doses of sodium hypochlorite:
0.44, 0.72, 0.86, 1.00, 1.50 and 2.00 mg/L. Sodium dichloroisocyanurate was added at concentrations of 200 and 400 mg/L. Ozonation
was applied at 16, 95, 127, 174, 237, 300, 348, 396 and 459 mg/L
with an ozone generator that produces 950 mgO3/h. The hydrogen
peroxide dosages were: 2.6%, 3.8%, 5.0% and 10.0% v/v. Finally, the
sterilization by pasteurization was carried out at temperatures of
40, 60, 70 and 90 C for 5 min.
2.2. Evaluation of sterilization methods
The effectiveness of each sterilization method was checked by
following the growth of bacteria in the 100 mL Erlenmeyer asks
each day (during 7 days). The initial pH of each sample was adjusted to pH 7.8 to allow a comparison among asks starting at
the same conditions. As estimation, the bacterial contamination
was measured spectrophotometrically by the absorbance at
680 nm (Helios Omega UVVIS Spectrophotometer, Thermo Scientic, Horsham, England) as described by Brown (1980).
The asks which did not seem to present any contamination
were checked by a bacterial count test as described by Eaton
et al. (1995). This method was also used for the supernatant obtained after harvesting microalgae cultures and for seawater culture medium in order to check the effectiveness of the
sterilization. It is also included water obtained from a ltration
unit installed in the Estacin Experimental Fundacin Cajamar
which consists of several lters of different pore size lasting in a lter of 1.2 lm (Merck Millipore, Germany).
To carry out the bacterial count 0.1 mL aliquots of the samples
were placed in Petri dishes (each sample in triplicate). Each sample
was previously diluted (1:10, 1:100 or 1:1000 or 1:10,000) because
of the different bacteria load in each one. The Petri dishes

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C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

contained a culture medium composed of tryptic soy agar 1% w/v

in seawater (TSA; Hardy, Santa Maria, CA, USA) as it is widely recommended for water and wastewater applications (Eaton et al.,
1995). The samples were incubated for 48 h at 25 C. After that,
the grown bacteria colonies were counted to determine the concentration in CFU/mL.
2.3. Microorganism and culture conditions
The strain Nannochloropsis gaditana B-3 was obtained from the
Marine Culture Collection of the Institute of Marine Sciences of
Andaluca (CSIC, Cdiz, Spain). The stock cultures were maintained
photoautotrophically in 100 mL Erlenmeyer asks containing
50 mL of culture. They were aseptically grown on an orbital shaker
at 100 rpm without aeration under a continuous illumination of
100 lE/m2/s and a temperature of 25 C. The seawater culture
medium used was the commercial medium Algal (Bionova, Santiago de Compostela, Spain) prepared at 8 mM concentration of
potassium nitrate. The inorganic components were added to natural seawater. The macronutrients were sterilized in autoclave
(Presoclave-II, J.P. Selecta, Barcelona, Spain) at 126 C for 20 min
and the micronutrients were ltered (Whatman GF/F 47 mm, nominal pore size 0.22 lm, Maidstone, United Kingdom).
The recirculated supernatant used for the tests was obtained
from outdoor cultures in tubular photobioreactors (Estacin Experimental Fundacin Cajamar). These reactors were operated in continuous mode as described by Acin et al. (2012) harvesting the
microalgae culture with a disc centrifuge (Westfalia OTC3, Holland
at 268 L/h).
2.4. Effect of the sterilization methods in N. gaditana growth
N. gaditana cultures were tested in order to check if the microalga is able to grow correctly using a recirculated supernatant (obtained after centrifugation of the cultures of the Estacin
Experimental) or if it is necessary to mix this supernatant prior
to the recirculation with new culture medium (Algal) and in what
extent. For this, experiments in (i) batch mode and (ii) continuous
modes were carried out.
2.4.1. Batch mode
With regard to the batch mode 100 mL Erlenmeyer asks were
used in order to check the effect on N. gaditana growth under the
temperature and irradiance previously described. The reported
data are mean values of nine experimental measurements.
In this sense, a second series of batch experiments in 100 mL
Erlenmeyer asks were carried out to select the proportion of the
supernatant that is possible to recirculate. Therefore, the medium
supplied to the microalgae cultures consisted of mixtures of supernatant and new seawater medium in the following proportions: 0
100, 2575, 5050, 7525 and 1000 (supernatant-Algal medium).
An additional test consisted of 100% recirculated water but replenishing the nutrients consumed during the culture (1000). The
tests were carried out in triplicate.
2.4.2. Continuous mode
After the batch tests, experiments at larger scale (1.8 L bubble
column photobioreactors) were carried out indoors in continuous
mode in order to verify the tests previously performed at smaller
scale. The proportions of recirculated supernatant to Algal medium
were the same than the described for the batch tests: 0100, 50
50, 7525,1000 and 1000. The culture system consisted of
two bubble column photobioreactors (1.8 L capacity, 0.07 m diameter, 0.50 m height) jacketed for temperature control at 18 C. They
were bubbled with air at 0.5 v/v/min. The pH was controlled at 8.0

by on demand injection of carbon dioxide. The reactors were

articially illuminated simulating a solar cycle by six Phillips PL32 W/840/4p white-light lamps. The irradiance on the reactor surface was controlled by an automated system to provide a
maximum irradiance of 1000 lE/m2/s at noon. The dilution rate
was xed at 0.3 1/d. The steady states attained were maintained
at least for three days to completely ensure steady conditions
regarding the biochemical composition of the biomass. Three samples were measured each day on the last three days of every steady
state; therefore, the reported values are the means of nine
measurements.
For both operation modes the new seawater culture medium
(Algal) and the recirculated supernatant were sterilized using the
most successful method studied in this work, taking into account
the obtained results in Section 2.2 and Section 2.4.
The cultures were harvested by centrifugation (9100g, 5 min),
washed with a 0.5 M aqueous ammonium bicarbonate solution,
centrifuged again and freeze-dried. The dry biomass was analyzed
immediately or stored at 22 C for up to 10 days prior to analysis.
2.5. Analytical procedures
The microalgal biomass concentration was estimated spectrophotometrically by measuring the absorbance at 540 nm (Helios
Omega UVVIS Spectrophotometer, Thermo Scientic, Horsham,
England) and it was veried by dry weight measurements. The
physiological status of the cells was determined by the uorescence of chlorophylls (Fv/Fm) using a uorimeter (AquaPen-C APC 100, Photon Systems Instruments, Czech Republic). The presence
of organic matter in the recirculated supernatant was measured by
the total organic carbon concentration (TOC analyzer, Shimadzu VCPH, Japan).
The consumption of nutrients by the microalgae was analyzed
after ltration to eliminate particles (0.22 lm) following the Ofcial methods of analysis of AOAC (2002). Nitrates: by absorbance
at 220 and 275 nm in an acid environment. Phosphorous: by a colorimetric method with ammonium molybdate at 430 nm. Sulfates:
by turbidimetry. Iron, copper, manganese and zinc: by atomic
absorption (Perkin-Elmer Analyst100, EE UU).
The fatty acid content of the biomass was analyzed by gas chromatography as described by Rodrguez-Ruiz et al. (1998) using
freeze-dried biomass. The total lipid content was determined as
described by Kochert (1978). Ash and protein contents were analyzed by the methods described by Brown et al. (1989) and Lpez
et al. (2010), respectively. Finally, carbohydrates content was
determined by difference between 100 and the percentage of the
rest of the fractions (ashes, proteins and total lipids). The elemental
analysis of the biomass was performed by means of a LECO CHNS932 analyzer (St. Joseph, MI, USA).
Finally, the statistical analysis of the data was carried out using
the software Statgraphics version 7.0.
2.6. Kinetic parameters
The nutrient requirements of the biomass were determined
using the growth stoichiometry of the microalga by the following
equation:

aCO2 bKNO3 cH2 O ! Cw Hx Oy Nz dO2

CwHxOyNz represents the dry microalgal biomass. This equation

is presented from a macroscopic point of view and it only takes
into account the nutrients that could be limiting for the growth
of the microalga. To solve the equation one mol of biomass was
xed as the reference (w = 1). The elemental composition of the
biomass was determined in order to calculate the stoichiometric

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C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

where S and S0 are the nal and initial substrate concentrations (g/
L), respectively and X is the average biomass concentration (g/L) at
the stationary state.
The substrate requirement (YS/X, g substrate consumed/g biomass) is easily obtained as the inverse of substrate yield.
The kinetic parameter used to control the uptake of the substrate was the biomass specic uptake rate (g substrate/day/g biomass), which was calculated with the following equation where D
is the dilution rate (0.3 1/d).

coefcients. The biomass is composed of C, H, O and N in 90

95% d.w., so the atomic balances of these four elements (referred
to one atom of carbon) would allow determining the coefcients.
After this, this formula permitted to estimate the nutrient requirements of the microalgae. Then, the yield of consumption of a
nutrient was obtained from the Eq. (2) and the substrate yield
(g biomass/g substrate consumed) was determined by Eq. (3):

Y X=S

Molecular mass of the biomass

b  Molecular mass of substrate

Y S=X

S  S0
X

0.5

0.5
control
2 mg/L
1.5 mg/L
1 mg/L
0.86 mg/L
0.72 mg/L
0.44 mg/L

0.4

0.4

0.3

0.2

0.3

0.2

0.1

0.1

0.0

0.0
0

10

12

Time, d

Time, d
0.5

0.5
control
459 mg/L
396 mg/L
348 mg/L
300 mg/L
237 mg/L
174 mg/L
127 mg/L
95 mg/L
16 mg/L

0.4

0.3

control
10 %v/v
5 %v/v
3.8 %v/v
2.6 %v/v

Hydrogen peroxide

0.4

Absorbance, 680 nm

Ozone

Absorbance, 680 nm

control
200 mg/L
400 mg/L

Sodium Dichloroisocyanurate
Absorbance, 680 nm

Sodium hypochlorite
Absorbance, 680 nm

D  S  S0
X

qs

0.2

0.3

0.2

0.1

0.1

0.0

0.0
0

Time, d

Time, d
0.5
control
90 C
70 C
60 C
40 C

Pasteurization

Absorbance, 680 nm

0.4

0.3

0.2

0.1

0.0
0

10

Time, d
Fig. 1. Inuence of the sterilization method at different dosages and conditions in seawater culture medium (Algal medium). The rst number in abscises axis denotes
sterilization methods. 1: autoclave (control), 2: sodium hypochlorite, 3: sodium dichloroisocyanurate, 4: ozone, 5: hydrogen peroxide, 6: pasteurization.

434

C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

3. Results and discussion

Regarding the tests about the sterilization methods (Section 2.1), the culture medium was sterilized by different techniques in order to determine the optimal conditions. The
effectiveness of each method was rst estimated by the absorbance
of the sample at 680 nm during 7 days (Fig. 1). The control
consisted of a sample that was autoclaved at 126 C for 20 min to
ensure a complete sterilization. So the culture mediums presenting
absorbance values similar the control are considered appropriate
for sterilizing. Chlorination seems to be effective at sodium hypochlorite concentrations over 0.88 mg/L. The procedure based on
adding sodium dichloroisocyanurate sterilizes the medium at a
concentration of 200 mg/L as recommended the Milton method,
used to sterilize materials for babies. Ozonation eliminates contaminants even at the lowest tested concentration, 16 mg/L. The
addition of hydrogen peroxide sterilizes the medium at dosages
above 5% v/v. Finally, pasteurization is effective at temperatures
over 60 C (5 min treatment).
However, the absorbance measurement is not enough as a
method to assess the quality of the sterilization. Because of that,
the bacterial count has been used. The analysis made to the seawater culture medium showed a low bacterial load of 3.7104 CFUs/

mL (Fig. 2a). If this medium is ltered at 1.2 lm the bacterial load

is reduced 33%, up to 2.5 104 CFUs/mL. Lastly, if the culture medium is ozonizated at 95 mg/L it does not show signicant levels
of contamination (2.0102 CFUs/mL) as the bacterial load is reduced
in two orders of magnitude. The same analysis was applied to the
recirculated supernatant obtained after harvesting the microalgal
cultures and similar results were obtained (Fig. 2b). Thus, the initial bacterial load of the supernatant is high (1.8106 CFUs/mL)
due to the recirculation of bacteria and organic matter that promote their development. If the supernatant is ltered the bacterial
load is reduced only one order of magnitude (1.9105 CFUs/mL).
However, when using ozonation it is possible to reach a reduction
of three orders of magnitude (1.9103 CFUs/mL). It ozonation decreases the bacterial load 1000-fold and 10-fold with regard to
the recirculated supernatant and the initial seawater medium,
respectively. A statistic study was performed to show the mean
values of contamination reached for each tested sterilization method and the intervals around each mean value. The method used to
discriminate among the mean values was the Fishers Least Significant Difference (LSD) procedure. The condence bars of these
methods overlap, which means that all could be used without signicative differences (Fig. 2a, similar methods noted with the same
letter). This allows observing that there are signicant differences

2.0e+6

2.0e+6

(a)

Algal medium

1.0e+6
5.0e+5

4.0e+4
3.0e+4

1.5e+6

Bacteria load, CFUs/mL

2.0e+4

1.0e+6
5.0e+5

4.0e+4
3.0e+4
2.0e+4
1.0e+4

1.0e+4

6.
90
C

5.
5

g/
L

g/
L
4.
95

g/
L
m

4.
95

6.
90
C

5.
5

g/
L

g/
L
m

g/
L
3.
20
0

2.
1

tio
n
Fi
ltr
a
1.

In
itia
l

0.0

0.0
3.
20
0

2.
1

1.
F

In
iti
al

Sterilization method

Sterilization method
2.0e+6

2.0e+6

(c)

Pasteurization temperature, C

bc

0.0

b
4.
19
0

6.
60

6.
40

In
itia
l

6.
90

6.
70

a
0.0

5.0e+5

4.
95

5.0e+5

1.0e+6

4.
47

1.0e+6

1.5e+6

In
iti
al

1.5e+6

(d)

Recirculated supernatant
Bacteria load, CFUs/mL

Recirculated supernatant
Bacteria load, CFUs/mL

(b)

Recirculated supernatant

iltr
at
io
n

Bacteria load, CFUs/mL

1.5e+6

Ozone concentration, mg/L

Fig. 2. Variation of bacterial load with the sterilization method used in Algal medium and recirculated supernatant (a, b). Inuence of different temperatures: 40, 60, 70 and
90 C for 5 min used in pasteurization (c). Ozonation: 2.5, 5, 10 min of exposition time (d). The rst number in abscises axis denotes sterilization methods. 1: ltration
(control), 2: sodium hypochlorite, 3: sodium dichloroisocyanurate, 4: ozone, 5: hydrogen peroxide, 6: pasteurization. Note, for example, that 5 min of ozone exposition
corresponds to 95 mg O3/L. All the following gures result from a Multifactorial ANOVA Statistical Study (mean values with different small letters denote signicant
difference, P < 0.05).

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C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

1.0

Biomass concentration, g/L

aa

a a

0.8

d d
0.6

ee

0.4

b b
bb
0.2

cc

c
c

1.
f

ilt
ra
t
2. ion
2
m
2.
g/
1.
L
5
m
2. g/L
1
m
2.
g
0.
86 /L
m
3.
20 g/L
0
m
g
4.
96 /L
m
4.
g
/L
16
0
m
4.
32 g/L
0
m
4.
48 g/L
0
m
g
5.
10 /L
%
5. v/v
5
%
v
6. /v
60

6. C
90
C

0.0

Used Method
Fig. 3. Inuence of the sterilization method on the biomass concentration of N.
gaditana. Error bars correspond to mean values of biomass concentration in
stationary state for each method. 1: ltration (control), 2: sodium hypochlorite, 3:
sodium dichloroisocyanurate, 4: ozone, 5: hydrogen peroxide, 6: pasteurization. All
the following gures result from a Multifactorial ANOVA Statistical Study (mean
values with different small letters denote signicant difference, P < 0.05).

between the group of methods 2, 3, 4, 5 and 6 (sodium hypochlorite, dichloroisocyanurate, ozone, hydrogen peroxide and pasteurization) and the ltration (1). Similarly Fig. 2b shows that all
methods of sterilization are effective for sterilizing the recirculated
supernatant with the exception of ltration. It was expected that
ltration would not cause a sharp decline in bacterial load because
the pore size employed was 1.2 lm, which is high. The results
show that with pasteurization (Fig. 2c) the best results were
achieved at 90 C (5 min). However, the most remarkable fact is
the high effectiveness of the ozonation (Fig. 2d) at a concentration
of 95 mg/L obtained with a short contact time (5 min) resulting in a
complete reduction of the bacterial load. Other chemical methods
such as the addition of hydrogen peroxide or sodium dichloroisocyanurate resulted in chemical alteration of the water, observed as
changes in color, especially when using dichloroisocyanurate.
To select the best method to reduce the bacterial load in the
recirculated supernatant it is also important to pay attention to
its inuence on the microalga growth. Therefore, laboratory scale
batch cultures of N. gaditana using the supernatant obtained after
harvesting the microalgae have been performed (see Section 2.4).
The aim is to check if the use of these sterilization methods involves a harmful effect on the growth of the microalga. For that,
the growth of Nannochloropsis is followed as shown in Fig. 3. It is
observed that, although several methods are able to sterilize in
any extent the supernatant (2, 3, 4, 5 and 6), only ltration and
ozonation are tolerated by the microalgae. It can be seen how only

the asks grown with the ozonized supernatant at low concentration (95 mg/L and 160 mg/L) and the ltered one are not affected
by the sterilization method attaining similar biomass concentration values (0.8 g/L). The statistical study reveals that these two
methods do not show signicant differences. Nevertheless,
Fig. 2a and b shows that ltration did not reduce the bacteria load
to suitable values. Kawachi and Nol (2005) reported that this procedure is inefcient killing the most resistant spores, although it
does kill most life in the water. Regarding the other sterilizing
methods it was observed that all of them affected negatively the
growth of the N. gaditana. The chemical methods probably alter
the composition of the medium and the pasteurization could oxidize compounds due to the high reached temperatures (usually
95 C). These changes in the medium composition could inuence
the microalga metabolism.
In aquaculture it is usual to have large tanks containing seawater. In that case, ash sterilization is commonly carried out using a
titanium plate heat exchanger that could be quite expensive. On
the other hand, an ozone generator can also be costly but the ozone
is able to achieve complete bacteria elimination in contrast to ltration. Accordingly, ozonation seems to be the most adequate
method to be used for the sterilization of mediums to grow
microalgae.
As ozonation seems to be the best sterilizing method, the total
organic carbon concentration (TOC) of the samples treated by
ozonation and ltration was analyzed in order to compare them
(Table 1). The results obtained show that the organic matter in
the recirculated supernatant reaches a value of TOC of 73.7 mg/L.
This organic matter is mostly composed of cellular fragments and
to a lesser extent of other dissolved organic compounds. The dissolved organic compounds are those that are potentially rst oxidized and degraded in the presence of strong oxidizing agents
such as ozone. By this way, the application of 95 mg/L of ozone
to the sample reduces the TOC in 33.5%. Applying higher concentrations of ozone it is possible to achieve higher TOC depurations,
but it would affect the microalga growth, as previously described.
When the sample is just ltered at 1.2 lm the TOC depuration
reaches 50.4%, which is quite higher. If the ltered sample is afterwards ozonized it is possible to increase the TOC depuration up to
72.1% but this requires a very high ozone dosage (3133 mg/L), so
this process would show low effectiveness. It is possible to assess
that ltration is not enough to ensure a properly sterilization of
the medium in spite of these tests and taking into account the bacteria load measured in the previous experiments carried out. Once
the best sterilization method has been chosen it is necessary to
evaluate the behavior of the cultures of N. gaditana growing in
the ozonized medium. They were rst grown in batch mode in
100 mL asks using the ozonized recirculated supernatant as the
culture medium. No negative effect on the growth was observed
(Fig. 4a) as the obtained biomass productivities were over the
one attained in the control ask, which used Algal medium (0
100). The higher the proportion of supernatant used, the higher
the biomass concentration, reaching values from 25% to 31% higher
than in the control ask in the best case. That demonstrated that

Table 1
Results obtained from the recirculated supernatant treated with ozone and ltration to reduce the dissolved organic carbon.
Treatment

Initial
without
treatment

Ozone
16 mg/L

Ozone
95 mg/l

Ozone
197 mg/L

Filtration
1.2 lm

Filtration
1.2 lm + Ozone
16 mg/L

Filtration
1.2 lm + Ozone
95 mg/L

Filtration
1.2 lm + Ozone
197 mg/L

Filtration
1.2 lm + Ozone
3133 mg/L

TOC; mg/L
Depuration,
%

73.7 2.2
0.0

71.0 2.1
3.8

49.0 1.5
33.5

43.9 1.3
40.5

36.6 1.1
50.4

28.0 0.8
62.0

27.4 0.8
62.8

27.3 0.8
62.9

20.6 0.6
72.1

C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

0.12

(a)

Batch cultivation

YX/S

3.0

1.5

YS/X

(a)

YX/S, g biomass/g nitrates

Biomass Productivity, g/L/d

0.10

0.08

0.06

0.04

2.5

2.0

1.0

1.5

1.0

0.5

0.5

0.02

0.0

0.00

0.0
0-100

0-100

25-75

50-50

75-25

100-0

50-50

75-25

100-0

100*-0

100*-0

Recirculated supernatant-algal medium

0-100
50-50
75-25
100-0
100*-0

0.06

1.0

(b)

Continuous cultivation 0.3 1/d

(b)
b)

0.05

0.8
0.04

qS, g/d/g

Biomass Productivity, g/L/d

YS/X, g nitrates/g biomass

436

0.6

0.03

0.02

0.4

0.01
0.2
0.00
Nitrates

Phosphates

K+

Mg+2

Sulphates

0.0

0-100

50-50

75-25

100-0

100*-0

the recirculated supernatant was still rich in nutrients that could

be reused by N. gaditana or even it could consume organic matter
dissolved in the supernatant. However, if the lacking nutrients of
the recirculated supernatant are replenished (1000) the biomass
concentration is 15% higher than if it is not replenished (1000).
The bacteria load was measured in all the cultures obtaining very
low values, below 1000 CFUs/mL.
Microalgae production plants usually operate in continuous
mode. During the culture large amounts of water and nutrients
are discarded. Therefore, it is necessary to design a closed system
to recirculate the medium making possible to use the nutrients
not ingested by the microalgae, as a strategy for water and nutrients saving which would involve a reduction in costs. Accordingly,
after the batch cultures in asks, 1.8 L bubble column photobioreactors were inoculated with N. gaditana and batch cultures were
performed to have a concentration of biomass of 1 g/L. Then, the
continuous culture was carried out at a dilution rate of 0.3 1/d.

Biochemical composition, % d.w.

Fig. 4. Mean values of biomass productivity (g/L d) of cultures tested at different

percentages of recirculated supernatant in (a) batch culture (0100, 2575, 5050,
7525, 1000, 1000 and (b) continuous culture at a dilution rate of 0.3 1/d (0
100, 5050, 7525, 1000, 1000).

Ash
Carbohidrate
Protein
Lipid

60

Recirculated supernatant-algal medium

50

(c)
c)

40

30

20

10

0
0-100

50-50

75-25

100-0

100*-0

Fig. 5. Nutrient yield (g biomass/g nitrates) and nutrient requirements (g nitrates/g

biomass) for all tests carried out in continuous mode (0100, 5050, 7525, 1000,
1000) (a). Biomass-specic uptake rate (g/d/g) of key inorganic compounds (NO
3,
2
+
+2
PO3
4 , K , Mg , SO4 ) for all tests (b). Biochemical composition for all tests (c).

using mixtures of new seawater culture medium (Algal medium)

and recirculated supernatant at different proportions (see Section 2.4.2). The obtained results (Fig. 4b) show how N. gaditana
can properly grow using a mixture 5050 of supernatant and Algal

Table 2
Empirical formula of the biomass obtained in the steady state of the continuous cultures of N. gaditana at 0.3 1/d using different mixtures of recirculated supernatant and Algal
medium.
Test

0100

5050

7525

1000

1000

Empirical formula

C1H1.36N0.14O0.51

C1H1.05N0.14O0.51

C1H1.19N0.11O0.50

C1H1.22N0.09O0.42

C1H1.39N0.10O0.68

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C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

Table 3
Fatty acids prole expressed as percentage on a biomass dry weight basis for the continuous cultures of N. gaditana at 0.3 1/d dilution rate using different mixtures of recirculated
supernatant and Algal medium. Note: N.I.: not identied.
Fatty acid

14:0
16:0
16:1n7
18:0
18:1n9
18:2n6
20:4n6
20:5n3
N.I.
Total

Test
0100

5050

7525

1000

100-0

1.2310.047
3.9020.150
3.4420.132
0.1400.005
0.8610.033
0.4110.016
0.7920.030
2.7020.104
2.3330.089
15.8650.608

0.6650.033
4.7120.236
4.8160.241
0.1050.005
0.6450.032
0.2280.011
0.7440.037
3.1400.157
1.7590.088
18.1890.909

1.0970.055
6.9220.346
6.7720.309
0.1060.005
0.8780.044
0.5440.027
1.1220.056
3.3730.199
2.3310.117
23.1451.157

1.0610.053
8.0510.403
6.9600.348
0.2110.011
1.3280.066
0.3780.019
0.9560.048
3.4400.172
2.0180.101
25.6221.281

1.5430.027
5.7550.102
4.4300.078
0.1950.003
1.2190.022
0.4620.008
0.9290.016
2.7070.048
2.4660.044
19.7070.348

medium reaching a biomass productivity near 0.8 g/L/d. Nevertheless, when the ratio of supernatant recirculation is increased (75
25 and 1000) the biomass productivity decreases to values of
0.4 g/L/d mainly because of the lack of nutrients in the medium.
There are hardly any difference between the tests 7525 and
1000 probably because of the lack of any essential nutrient to
the growth in both cases. On the other hand, when using only
the recirculated supernatant after the replenishment of the consumed nutrients (1000) it was possible to achieve a biomass productivity near 0.8 g/L/d, as the one obtained with Algal medium
(0100) with no statistically signicant differences between them.
The use of mixtures of recirculated supernatant and Algal medium
50:50 v/v or the use of only recirculated supernatant after the
replenishment of the lacking nutrients lead to a reduction of 50%
or 100%, respectively, in water usage (water evaporation and water
loss in harvesting are not considered). It also leads to a reduction in
the addition of nutrients up to 50% or 15%, respectively.
The elemental composition of the microalgal biomass harvested
in the steady state of the continuous cultures was analyzed in order to establish the empirical formula (Table 2). This formula is
quite similar whatever the composition of the culture medium
used. When using pure Algal medium the obtained formula is
C1H1.36N0.14O0.51. From the elemental analysis reported by Pan
et al. (2010) it is possible to determine an empirical formula for
Nannochloropsis sp. of C1H1.63N0.08O0.40, which is analogous to the
one obtained in this work. Having established the empirical formula it is possible to calculate the nitrate yield (Y X=S ) and the nitrate requirement (Y S=X ). It was calculated for the different ratios
of recirculated supernatant and Algal medium (0100,
5050, 7525, 1000 and 1000). It was shown (Fig. 5a) that the
higher the ratio of recirculated supernatant to Algal medium the
higher the yield coefcient for nitrate (YX/S) and the lower the
requirement of nitrate (YS/X). This would lead to an increase in
the content in lipids and a decrease in proteins. However, with
the recirculated supernatant after replenishing the nutrients
(1000) is more than 30% lower than the obtained when using Algal medium (0100).
The biomass-specic uptake rates of key inorganic nutrients,
3
2
+
+2
such as NO
3 , PO4 , K , Mg , and SO4 , was determined in order
to compare them (Fig. 5b). As shown, the recirculation of the
supernatant can signicantly reduce the uptake rate of nutrients.
3
+
The major usages correspond to NO
3 , PO4 and K with values of
0.049, 0.017 and 0.014 g/d/g, respectively. In the test with the
replenished supernatant (1000) the uptake rate of nitrate decreases in 50% with regard to the test with Algal medium. Nevertheless, the rate for the uptake of the other inorganic nutrients
remains constant. The reduction of nutrients consumption (up to
50%) is not noteworthy in the context of aquafeeds as biomass production for this purpose is not large. Otherwise, this reduction
would have an important impact with regard to the sustainability
of the processes related to the production of commodities.

The biochemical composition of the biomass was analyzed to

check if the use of the recirculated supernatant had a negative or
a positive effect on the biomass quality (Fig. 5c). Total lipids, proteins, carbohydrates and ashes were measured for the biomass of
the steady state of the continuous cultures. Moreover, the fatty
acids prole was determined (Table 3). The biomass was composed
of a higher amount of total fatty acids when employing the mediums 5050 and 1000 compared to the one of the control (Algal
medium, 0100). In particular, it had 96% and 26% more fatty acids
for the tests at 5050 and 1000, respectively, than for the test at
0100, attaining a maximum value of 30.2% d.w. for the 5050 test.
That was accompanied by an increase in the amount of reserve lipids such as 14:0, 16:0, 16:1n7 and 18:1n9. The content in structural lipids (as 20:4n6 and 20:5n3) did not change. The content
in proteins of the biomass was similar for the test at 0100 and
1000, but it was lower for the one at 5050 because the metabolism of the microalgae was more displaced to the production of
lipids due to the lack of nutrients. The same tendency was observed regarding the carbohydrates content (Fig. 5c). Therefore,
the lack of nutrients (test 5050 or 1000) leads to an increase
of 50% or 100%, respectively in total lipids and a decrease in the
amount of ashes. That is in accordance with the accumulation of
fatty acids by carbon availability in excess (Roessler, 1990; Tan
and Johns, 1996; Wen and Chen, 2000). Rodol et al. (2003) assess
that the recirculation of the medium reduces the biomass productivity in 50% and favors its contamination. On the other hand, the
biomass productivity obtained after the recirculation of the medium in this work is similar to the achieved using Algal medium.
Moreover, the biomass is of a high quality for aquaculture because
of its high content in proteins and lipids (particularly, EPA).

4. Conclusions
If mass production of microalgae is ever to come about it is necessary that the culture medium be reused. This work has shown
that the supernatant obtained from harvested N. gaditana cultures
can be reused after ozonation at 95 mgO3/L as this ensures a proper
sterilization. It will allow reducing the nutrient costs, in a extent
we expect to be able to quantify in future contributions, once the
method is scaled-up. It has also been shown that N. gaditana tolerates the recirculation of the medium replenishing the depleted
nutrients, and the obtained microalgal biomass is of high quality,
suitable for aquaculture because of its high content in proteins
(50%) and lipids (40%) with more than 3% EPA (d.w.).

Acknowledgements
This research was supported by the General Secretariat of Universities, Research and Technology of Andalusian Government

438

C.V. Gonzlez-Lpez et al. / Bioresource Technology 129 (2013) 430438

(AGR-5334) and was co-nanced by FEDER funds. We would also

like to thank the support of Fundacin CAJAMAR.
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