Algal Research 16 (2016) 167176

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Algal Research

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

Microalgae biomass production using wastewater: Treatment and costs

Scale-up considerations
Lusa Gouveia a, Soa Graa a, Catarina Sousa a, Lucas Ambrosano b, Belina Ribeiro a, Elberis P. Botrel b,
Pedro Castro Neto b, Ana F. Ferreira c,, Carla M. Silva d
a
LNEG Laboratrio Nacional de Energia e Geologia, I.P./Bioenergy Unit, Estrada do Pao do Lumiar 22, 1649-038 Lisboa, Portugal
b
Universidade Federal de Lavras, Laboratrio de Pesquisa em leos, Gorduras e Biodiesel, Lavras, Brazil
c
IDMEC, Instituto superior Tcnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal
d
IDL Instituto Dom Luiz, Departamento de Engenharia Geogrca, Geofsica e Energia, Faculdade de Cincias, Universidade de Lisboa, Portugal

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

Article history: This work is part of a LIFE project to treat urban wastewater from guas da Figueira (AdF, Figueira da Foz, PT)
Received 30 November 2015 using a vertical tubular photobioreactor (PBR) prototype (150 L), to be scaled up and integrated in a waste
Received in revised form 25 February 2016 water treatment plant (WWTP). The PBR was inoculated with three different microalgae: Chlorella vulgaris
Accepted 5 March 2016
(Cv), Scenedesmus obliquus (Sc) and Consortium C (ConsC), isolated from the efuent. The study intends to nd
Available online xxxx
the best microalga in terms of wastewater remediation, biomass productivity and quality, for further uses,
Keywords:
such as biofuel, biofertilizer and bioplastic production.
Bubble column photobioreactor The experiments achieved volumetric productivities of 0.1 g/Ld (Cv), 0.4 g/Ld (Sc) and 0.9 g/Ld (ConsC). The
Wastewater treatment maximum removals attained by Cv, Sc and ConsC were: 84, 95 and 98% for total nitrogen; 95, 92 and 100% for
Electrocoagulation phosphorus; and 36, 63 and 64% for COD, respectively. The treated water had values that are in accordance
Solar drying with environmental legislation (Directive 98/15/CE).
Scale-up Electrocoagulation was tested and resulted in an energy saving of 89%, compared with centrifugation alone. For
drying the biomass, a solar dryer was used. Costs of overall processes versus conventional technologies are
discussed and compared with other facilities and target values.
2016 Elsevier B.V. All rights reserved.

1. Introduction The use of algae for wastewater treatment guaranteed remarkable

The global water crisis is mainly due to population growth in areas
with low freshwater resources, pollution of both surface and ground-
water, and long-term changes in the hydrological cycle due to climate
change [1]. Wastewater recovery is crucial for the better management
of water resources and can help the mitigation of regional or seasonal
water scarcity [1].
Presently, the treatment of the wastewater treatment plants
(WWTP) required a large amount of chemicals, and the operation and
maintenance of the technologies used are energy demanding processes
[2]. Both drawbacks reduced the environmental and energy sustainabil-
ity of the WWTPs.
In order to reduce the carbon footprint of these plants, other poten-
tial uses for wastewater included: (1) residential irrigation with reused
water [3,4], (2) land fertilization using the digested sludge [5,6] and
(3) possible production onsite of combined heat and power systems
(CHPs) [7].
Corresponding author.
E-mail address: lipa.ferreira@tecnico.ulisboa.pt (A.F. Ferreira).
advantages, such as: (1) the oxygen needed for the bacteria is provided
through microalgae photosynthesis, avoiding aeration thus, reducing
energy demand [8], (2) reduction in hazardous solid sludge formation
(e.g., heavy metals andpathogens) [9] (3) reduction of GHG emissions,
(4) reduced costs [9], and (5) the production of useful algal
biomassenergy rich recycling of the nutrients present in the wastewa-
ter. Fortier and Sturm [10] also claimed that a promising solution to re-
duce the freshwater and fertilizer demand of algal biomass production
is to utilize municipal wastewater efuent, which contains nitrogen,
phosphorus, and other necessary nutrients.
Nevertheless, as the microalgae are too small, cultures are much dilut-
ed, it is necessary to spend a lot of energy to recover the biomass, which
corresponds to a high percentage of the total production costs (30%)
[11]. Although centrifugation is an effective harvesting method, it pre-
sents high investment and operational costs. The electrocoagulation
method, a non-conventional technique for harvesting microalgae, was
studied in detail for Nannochloropsis sp. by the authors [12]. They obtained
the best recovery (N97%) using a current density of 8.3 mAcm2 for
10 min without signicant changes in the quality of the biomass both in
terms of fatty acid and pigment proles.

http://dx.doi.org/10.1016/j.algal.2016.03.010
2211-9264/ 2016 Elsevier B.V. All rights reserved.
168 L. Gouveia et al. / Algal Research 16 (2016) 167176
Other studies supported the relevant EC application in microalgae
harvesting and its importance towards commercial applications
(e.g., [1319]). NAABB [20] demonstrated that the use of EC using avail-
able commercial equipment could provide a 14% cost reduction.
The microalgae biomass is typically recovered by drying using an
oven, which consumes electricity from the grid. In order to overcome
the high energy consumption of this technique, solar drying systems
have been developed both for agriculture and forest products, although
these systems become seasonal. Nowadays, there is a lack of information
about the use of solar based energy for the production of natural products,
mainly algae-based products. However, the use of renewable energy is
relevant in the scale-up alternative processes when compared with con-
ventional ones based on fossil fuel sources of energy. Electrocoagulation
and solar drying seems to be potential future trends. [21].
The promotion of the rational use of energy and renewable energy
sources in the industrial sector has been thoroughly proposed by
European [22] and Portuguese programmes [23].
This biomass can be further processed to make biofuels or other
valuable products such as biofertilizers, biopolymers and biolms. It
can also be burned to produce heat and electricity.
According to Brennan [24], the conjunction of wastewater and biofu-
el production is probably one of the most economically and environ-
mentally sustainable ways to produce bio-energy and bio-products.
In this work all the cheapest and low consuming energy technolo-
gies/methodologies for making algal biomass production economically
feasible for several purposes were used, namely for biofuel and clean
water production.
An urban wastewater was treated with different microalgae
(Chlorella vulgaris (Cv), Scenedesmus obliquus (Sc) and Consortium C
(ConsC, isolated from the wastewater), using a vertical tubular
photobioreactor (PBR) (150 L). Some culture parameters (e.g., pH, air
agitation, recirculation ow) were optimized in order to treat the
wastewater and enhance biomass productivity. The other parameters,
such as dissolved O2, dissolved CO2, ammonium, temperature and
solar irradiation, were monitored along with the experiments. The ef-
ciency of the wastewater treatment (in terms of N, P, COD removals),
biomass productivities and the composition of the biomass were com-
pared between the species.
2. Material and methods
2.1. Efuent
The microalgae culture medium in the 150 L PBR was an urban
wastewater from guas da Figueira (AdF, Figueira da Foz, PT) collected
after primary treatment where the efuent has nutrients (particularly,
nitrogen and phosphorus) needed for microalgae growth.
During the time of experiments several campaigns were done to col-
lect and characterized the urban wastewater, namely from September
to November (Table 1).
2.2. Photobioreactor
The experiments were carried out at LNEG's Alfragide Campus,
Lisbon, located on the western coast of Portugal (384354.3N 912
41.3W) using a vertical prototype photobioreactor bubble column
(PBR) (150 L). The PBR, placed outdoor, has an air compressor to per-
form agitation, a membrane module to permeate the treated
Table 1
Urban wastewater composition of the different campaigns (after primary treatment).
Campaign pH EC (mS/cm) NH+
4 (mg/L) NO
3 (mg/L)
September 8.21 1.13 147.2 3.8
October 8.33 2.45 222.2 b2.2
November 8.34 2.43 473.0 b2.2
wastewater and a settler to concentrate the biomass (Fig. 1a). The PBR
included 12 poly(methyl methacrylate) tubes placed vertically, with a
diameter and height of 100 and 2000 mm, respectively (Fig. 1b). The
air compressor has a 2.72 kW power and recirculation pump of 0.37 kW.
The bioreactor operated in fed-batch mode. The average air temper-
atures were 23 C (September), 18 C (October) and 11 C (November).
The insolation values were around 350 h (September), 225 h (October)
and 160 h (November) (Table 2).
During the experiments samples were collected daily from the PBR
to analyse the microalgae growth and the evolution of nutrients in the
wastewater. Thus the pH, conductivity, ammonium, nitrates, phospho-
rous and COD values were determined with the same methods per-
formed to the efuent (described below).
2.3. Microalgae
The microalgae used in this work were Chlorella vulgaris (INETI 58,
LNEG_UB, Portugal) (Cv), Scenedesmus obliquus (ACOI 204/07, Coimbra
University Algotec, Portugal) (Sc) and Consortium C (ConsC), isolated
from the wastewater.
2.3.1. Consortium C isolation
The wastewater was ltrated using a glass microber (Whatman,
USA) and re-suspended in synthetic medium (Bristol) [11]. Daily sam-
ples were taken for optical microscope observation. The culture asks
were incubated at a temperature of 25 2 C, luminosity of 100 E/
m2s and agitation of 130 rpm. The consortium of microalgae isolated
included different strains such as Chlorella, Chaetophora, Scenedesmus
and Navicula (ConsC).
All the microalgae used (Cv, Sc, ConsC) were cultivated, before inoc-
ulation, in a 10 L cylinder reactor, with the appropriate culture medium
for each strain [26], at 25 C, continuous light of 100 E/m2s (measured
by an Phywe Lux-Meter) and agitated by aeration of compressed l-
tered air at a ow rate of 1 v/v/min.
2.4. Photobioreactor operation
The trials on the PBR were conducted with urban wastewater after
primary treatment from AdF (240 L), without any supplementation.
The three different microalgae strains were tested. At the beginning of
the trials all the inoculations were performed similarly independently
of the microalga specie: the volume of microalga inoculum was calculat-
ed in order to obtain an initial concentration on the PBR around an OD
(540 nm) of 0.30. When the algal growth reached the stationary
phase, the culture was collected (30 L) plus permeate (after passing
through the membranes) (30 L) and new wastewater was feed to the
PBR (60 L), in a semi-continuous feeding mode.
2.5. Microalgae growth
Microalgae growth was evaluated daily by measuring optical density
at 540 nm (OD540) using a JASCO V-530 spectrometer (the samples
were diluted appropriately in order to ensure that the measured optical
density values were assessed within a range of 0.11), and ash free dry
weight (AFDW) (by ltered the samples through a Whatman GF/C
45 m lter).
N-NO
3 (mg/L) PO3
4 (mg/L) PPO3
4 (mg/L) P2O5 (mg/L) COD (mg/L)
0.9 11.4 3.8 8.6 101
b0.5 14.3 4.8 10.8 131
b0.5 22.8 7.5 17.0 147
 L. Gouveia et al. / Algal Research 16 (2016) 167176 169

Fig. 1. Scheme (a) and photograph (b) of a closed poly(methyl methacrylate) photobioreactor (150 L), placed outdoors (c) with a membrane module and (d) settler.

2.6. Culture parameters
The PBR was fed with urban wastewater from AdF, collected after
primary treatment (Table 1).
The experiments were done between September and November
(Table 2).
Air agitation and recirculation ows were adjusted manually. Air ag-
itation and recirculation ow was manually adjusted based on 28 years
of experience on PBRs. However, the value was around 1 v/v/min, for air
agitation, and the recirculation ow allowed a residence time of 2
6 days.
The results will be used to scale-up a similar PBR of 1500 L.
at 10,000 rpm (Avanti J25, Beckman) for 5 min. The dewatering was done
by using a solar fan device. Both methods were compared to the tradition-
al ones (centrifugation and oven at 80 C).
The use of electrocoagulation before centrifugation was tested as an
alternative for harvesting procedures (Fig. 2). Electrocoagulation was
conducted by using aluminium electrodes and a current density of
8.3 mAcm2 for 10 min. The concentrated biomass was then centri-
fuged. Both harvesting techniques were assessed to evaluate the
electrocoagulation potential and the consequent energy and cost
reductions.
2.8. Microalga dewatering

After harvesting, the microalga biomass was dried using a regular

2.7. Biomass harvesting oven and drying (U10, Memmert) at 80 C for 3 h.
At the same time, a small scale prototype of a solar heater SECMAD
The harvesting of the biomass was performed by electrocoagulation was designed with a 0.28 m3 product volume and 0.8 m2 solar collector
using aluminium electrodes according to [12] and nally was centrifuged apertures (Fig. 3) and was used to dry the biomass and was compared to
the oven-dried process. In this case no articial energy was consumed
Table 2 and the process was faster than an oven and/or freeze-dryer. The solar
Average temperature of air and month insolation during the experiments [25]. heater was capable of drying about 1 to 2 kg of microalgae biomass
from 85% to 5% moisture content in about 3 to 4 h operating with a
Cv Sc ConsC
small 20 W electric DC fan to force ventilation. Both dewatering tech-
Average temperature (C) 23 18 11 niques were assessed to evaluate the solar dryer potential and the con-
Month insolation (h) 350 225 160
sequent energy and cost reductions.
170 L. Gouveia et al. / Algal Research 16 (2016) 167176

Fig. 2. a) Schematic diagram and picture of the experimental set-up. (1) magnetic agitator; (2) microalga culture; (3) aluminium electrodes; (4) DC power source (Matos et al.[12]);
b) photo of the whole EC system during the process; c) after operation (comparison with the control, without EC).

2.9. Analysis 2.9.2. Supernatant after microalgae biomass recovery

2.9.1. Efuent
All the urban wastewater from guas da Figueira (AdF, PT) collected
along the campaigns were analysed and characterized in terms of pH,
ammonium, nitrates, phosphorous and COD.
The pH measurement was performed using a pH electrode (Crison),
and for the ammonium (NH+ 4 ) measurement an ion selective electrode
NH+ 4 (Crison) was used.
The nitrate determination (NO 3 ) was performed by the method Ni-
trate Cell Test 1.14542 (test kits from Spectroquant Merck) using a
HACH DR/2010 spectrophotometer; this test gives the results in mg/L
of NO
3 (nitrate) and NNO3 , NNO3 being the nitrogen present in
the nitrate.
A commercial kit for phosphorous determination has been used
(also called orthophosphate) with the Phosver 3 (ascorbic acid) method
using Powder Pillows (Spectrophotometer HACH DR/2010). This test
gives the results in mg/L of PO3 3
4 (phosphate), PPO4 and P2O5 (phos-
3
phorus pentoxide), PPO4 being the phosphorus presented in the
phosphate.
The COD determination was done with test kits from Hach Lange,
Hach 21258-51 (0 to 150 mg/L COD). 2 mL of the sample, which was l-
tered with 0.22 m lters (Millipore) and properly diluted, was mixed
with the reaction solution in the test tube and heated for 2 h at 150 C
in a thermoblock. After cooling down to room temperature the test
tube was introduced into the photometer (HACH DR/2010) and mea-
sured at 620 nm. The COD (mg/L) results are dened as the mgO2 con-
sumed per litre of sample under conditions of this procedure.
All the analyses (nitrogen, ammonium and nitrate, phosphorous and
COD) performed for the efuent were done similarly for the superna-
tant after microalgae biomass recovery, in order to evaluate the efcien-
cy of the treatment.
2.9.3. Microalgae biomass characterization
Previous to the biomass characterization in terms of lipids, sugar and
protein contents, microalgae were ground for 4 min at a speed of 25 s
using a Retsch ball mill model MM400. The biomass was character-
ized on a dry basis after the electrocoagulation process to recover the
biomass from the culture, and using a non-energy consumption device
for drying (solar).
2.9.3.1. Lipid contentfatty acid composition. Lipid extraction from
microalgae biomass was carried out in a Soxhlet apparatus using 1.0 g
of biomass and n-hexane as a solvent, for 6 h. The amount of total lipids
was determined gravimetrically. The lipid fraction composition in terms
of fatty acids was determined by gas chromatography (GC). Fatty acid
methyl esters were prepared based on EN ISO 5509 (boron triuoride
method) [27]. The obtained samples were analysed by gas chromatog-
raphy using a CP-3800 GC (Varian, USA) equipped with 30 m
SUPELCOWAX 10 capillary column (0.32 mm of internal diameter and
0.25 m of lm thickness). Injector (split 1:50) and detector (ame ion-
ization) temperatures were kept constant at 250 C [28]. The oven tem-
perature programme started at 220 C for 16 min, increased at 20 C/
min until 230 C and kept constant of this temperature for 4 min. Carrier
gas, He, was kept at a constant rate of 1.3 mL/min.

Fig. 3. a) Schematic diagram; b) photo of the solar dryer SECMAD.

 L. Gouveia et al. / Algal Research 16 (2016) 167176 171

Fatty acid composition was calculated as percentage of the total fatty the maximum absorbance of the samples [34].
acids present in the sample, determined from the peak areas. Fatty acid
content was calculated according to the European Standard EN 14103
AV f
[29]. Total Pigments % 2
E1%
1cm  m

2.9.3.2. Sugar content. The extraction of sugars from the microalgae bio-
mass was performed by the addition of H2SO4 (2 N) to 500 mg of dried
microalgae and autoclaving for 60 min at 121 C. Then the sample was
ltered through a 0.2 m lter [30]. The total sugar content was deter-
mined by the phenol-sulphuric reagent method [31].
To 1 mL of sample previously diluted (1:20) was added 1 mL of phe-
nol solution (5% w/v) and 5 mL of 96% sulphuric acid. It rested for 10 min
at room temperature and for 15 min in a cold water bath. Then it was
measured with a spectrophotometer at 490 nm. A calibration curve
was prepared using glucose standards and the results of total carbohy-
drates in the microalgae samples are expressed in terms of glucose
equivalents (Eq. 1):
where A is the absorbance (at the wavelength of maximum absorption),
V is the total volume of the pigment extract (mL), f is the dilution factor,
E1%
1 cm is the extinction coefcient and m the weight of the sample (g).
The extinction coefcient used was based on an average of the E1% 1 cm
of the carotenoids mainly found in microalgae, according to Gouveia
and Empis [35].
The carotenoids were identied by thin layer chromatography
(TLC), using a silica gel plate, which was previously heated in an oven
at 80 C for 1.5 h. The eluent consisted of a mixture of petroleum ether
4060 C:acetone:diethylamine in the ratio 10:4:1 (v/v/v), respectively.
2.10. Statistical analysis

glucose slope  A490 intercept  dilution factor r2
0:9947
where A490 is the absorbance at 490 nm and glucose concentration is
expressed in g/mL.
2.9.3.3. Protein content. The total nitrogen present in the microalgae bio-
mass was determined using a modied Kjeldahl method [32].
In a Kjeldahl tube 200 mg of dried microalgae, 5 mL of distilled water
and 50 mL of digestion solution (134 g K2SO4 + 650 mL H2O + 200 mL
H2SO4 + 2 g HgO/25 mL H2SO4 (6 N)) were added. It digested in a diges-
tive device (Buchi Digestion Unit K-424) for 4 h. Afterwards it was
placed in the distillation device (Buchi Distillation Unit K-350) for
6 min with the reagent sodium hydroxide plus sodium thiosulfate.
Thereafter 50 mL of boric acid indicator solution was added to the
distillate.
Then it was titrated with a stock solution of H2SO4 (0.02 N). Crude
protein was calculated by multiplying total nitrogen by the convention-
al conversion factor of 6.25 [33].
2.9.3.4. Pigment content. The mixture of 15 mg of grounded microalga,
2 mL of acetone and glass beads were added to a tube, which was
2 min in a vortex mixer and 5 min in an ice bath. The sample was
then centrifuged (8 min at 3900 rpm) and the liquid phase was re-
moved and stored in a separate tube. This procedure was repeated 5
times, till the liquid became colourless. The total liquid phase collected
was quantied and ltered.
Total pigments were quantied by spectrophotometry (Hitachi-
2000). Spectra were run between 380 and 700 nm. Calculations were
performed using the Beer Lambert equation (Eq. (2)) with a value of
215 L/(gcm) for the specic optical coefcient at the wavelength of
All the determinations were repeated at least two or three times and
1
results were evaluated using the SISVAR software [36] and the average
comparison ScottKnott (p b 0.05) test. In Table 7 the statistical analysis
was only performed for the means of the main fatty acids present
(C14:0, C16:0, C18:0, C18:1, C18:2, C18:3 and C22:0).
3. Results
3.1. Wastewater composition
The wastewater had different compositions along the campaigns
mainly due to the seasonal variation of Figueira da Foz's population
and climate, and falling rain uctuations, as can be seen in Table 1.
3.2. Photobioreactor performance
In order to perform the wastewater treatment using microalgae, the
150 L PBR was inoculated with primary efuent from AdF and different
species were tested: Cv, Sc and Consortium C.
3.2.1. Chlorella vulgaris
The trial using Chlorella vulgaris (Cv) was done in July and lasted
12 days. The maximum productivity achieved was 0.1 g/Ld (day
5) and the maximum removal rates were: total nitrogen 84% on day
11, phosphorus 95% on day 8 and COD 36% on day 4. The trial ended
when the algal growth reached the stationary phase.
3.2.2. Scenedesmus obliquus
Another trial was performed in October with the primary efuent
from AdF and the microalga Scenedesmus obliquus (Sc). This experiment
lasted 13 days and had one additional feed, after the initial inoculation,
with 50 L of primary efuent on day 8.

Table 3
Nutrient monitorization in PBR (start, after 5 days and after 13 days).

NH+
4 (mg/L)
NO
3 (mg/L)
PPO3
4 (mg/L) COD (mg/L)
1 2 3 1 2 3 1 2 3
Initial 5 days 13 days Initial 5 days 13 days Initial 5 days 13 days Initial1 5 days2 13 days3
b b a b b b a b a a a
Cv 119.3 50.3 37.6 14.0 9.0 4.6 8.3 2.0 3.0 108.6 112.0 145.3a
Sc 175.3b 92.0a 3.0b b2.2c 1.6b b2.2b 4.6a 1.6b 0.3b 81.6a 57.6 b 76.0 b
ConsC 346.6a 12.3c 10.6b 54.3a 99.0a 27.0a 5.3a 3.0a 1.2b 75.0a 110.6 a 91.0 b
Legislation4 10 50 10 150

Results are the average of 23 replications.

Means followed by the same letter in the column did not differ signicantly from each other, ScottKnott test, p b 0.05; F test, p b 0.05.
1
Initial PBR start point inoculated with urban wastewater and microalgae culture.
2
5 days PBR after ve days of start point.
3
13 days PBR after thirteen days of start point.
4
Legislation PT Decreto-Lei no. 236/98.
172 L. Gouveia et al. / Algal Research 16 (2016) 167176

Table 4 Table 6
Nutrient maximum removal rates. Microalgae biomass composition.

Maximum removal rates (%) Sc Cv ConsC

N P COD Crude protein (%) 32.7c 56.4a 51.6b

Total lipids (%) 8.1ns 10.0ns 13. 6ns
Cv 84b 95b 36b
Total sugars (%) 11.7c 27.7a 20. 6b
Sc 95a 92b 63a
Total Pigments (%) 1.2a 1.2a 0.9b
ConsC 98a 100a 64a
Total Ash (%) 14.1a 14.5a 7.4b
Results are the average of 23 replications.
Results are the average of 23 replications.
Means followed by the same letter in the column did not differ signicantly from each
Means followed by the same letter in a line did not differ signicantly from each other,
other, ScottKnott test, p b 0.05; F test, p b 0.05.
ScottKnott test, p b 0.05. ns = non signicant, F test, p b 0.05.

This trial achieved a maximum of productivity of 0.4 g/Ld on day 9, 3.3. Harvesting and dewatering
and the maximum removal rates were: total nitrogen 95%, phosphorus
92% both on day 13, and COD 63% on day 5. Once again, the trial ended The pre-concentration of the cultures was done by electro-
when the algal growth reached the stationary phase. coagulation using aluminium electrodes and a current density of
8.3 mAcm2 for 10 min, and the concentrated biomass was then cen-
trifuged. Electrocoagulation plus centrifugation saved 89% of energy
3.2.3. Consortium C when compared to the centrifugation alone.
The trial with Consortium C microalgae took place in November and The dewatering was done using both an oven and a solar fan device
lasted 103 days. In this trial the PBR was inoculated with the primary ef- prototype. Using the solar fan (which lasted the same period of time (3
uent from AdF and the alga Consortium C (isolated from the efuent). 4 h)) reduced about 86% of energy consumption.
In spite of the ConsC being theoretically more adapted to the wastewa-
ter as a culture medium, the lower air temperature and insolation of the
time schedule of the trial were unfavourable. So the experiment took 3.4. Microalgae biomass
much longer and had a semi-continuous feed mode (seven feeds after
the rst inoculation). Each feed included about 60 L of primary efuent The results obtained for the biochemical characterization of different
and had a recovery of around 30 L of concentrated culture, which oc- microalgae biomass after treating the efuent are presented in Table 6
curred on days 37, 47, 57, 62, 72, 83 and 93. Notice that, before feeding and are expressed in weight percentage (AFDW ash free dry weight).
and recovering the biomass, the membrane module was used with two Cv and Sc have an intermediate total ash content of around 14%,
objectives: (1) concentrate the biomass to recover (around 30 L) and while ConsC has a lower ash content (7.4%). These values are higher
(2) remove permeate (treated water) (around 30 L). In this trial, the than the ash content reported in Silva et al. [38] for the same species
maximum productivity achieved was 0.9 g/Ld on day 71, and the max- of microalgae.
imum removal rates were: total nitrogen 98% on day 36, phosphorus Cv and Sc presented relatively low lipid values, 10.0% and 8.1%, re-
100% on day 83 and COD 64% on day 12. spectively. Scientic literature reports lipid contents of up to 1322%
The results of the nutrient monitorization before and after treat-
ment, nutrient maximal removal rates and microalgae biomass produc-
Table 7
tivities are depicted in Tables 3, 4, and 5 respectively. Fatty acids present in Chlorella vulgaris (Cv), Scenedesmus obliquus (Sc), and Consortium C
According to Directive 98/15/CE, the removal rate for total nitrogen (ConsC) oil extracts.
should be between 70 and 80% of the initial concentration in the medi-
Fatty acids Cv Sc ConsC
um, and for phosphorus 80%. Table 2 showed that the mandatory re-
(% p/p) (% p/p)* (% p/p)*
movals were achieved, proving that this PBR is sufciently efcient to
C8:0 0.15 0.40 0.12
treat wastewater.
C10:0 0.07 0.27 0.04
Using the microalgae Cv and Sc as an inoculum to perform the waste- C11:0 0.21 n.d. 0.37
water treatment, revealed that both of the nutrient removals (nitrogen C12:0 0.05 n.d. 0.03
and phosphorus) are close to the ones found in the literature, where the C13:0 0.10 n.d. 0.09
authors achieved N90% for N and N 98% for P [37]. Despite that ConsC C14:0 0.61b 2.35a 0.41b
C14:1 2.39 n.d. 3.28
achieved the best nutrient removal rates, as well as the best productivity C15:0 0.65 n.d. 0.27
it took longer. The good results in terms of nutrient removals are expect- C15:1 0.12 n.d. 0.03
ed due to the fact that this consortium was isolated from the efuent C16:0 20.0b 36.9a 18.7b
and therefore the microalgae present were more acclimated than the C16:1 1.39 1.93 0.45
C17:0 0.43 1.23 0.21
others. The longer time needed by ConsC was probably due to the
C17:1 0.31 n.d. 0.10
unfavourable conditions (lower air temperature and insolation). C18:0 1.32b 5.80a 1.31b
C18:1 13.3b 18.9a 11.9b
C18:2 12.9a 5.48b 13.1a
C18:3 22.8a 6.65b 19.1a
C20:0 1.19 n.d. 2.37
Table 5 C20:1 0.30 n.d. 0.59
Microalgae biomass productivities. C20:2 n.d. n.d. 0.14
C20:4 0.82 n.d. 0.16
Productivity (max) Productivity (average)
C22:0 0.52b 13.3a n.d.
(g/Ld) (g/Ld)
C24:0 n.d n.d. 0.19
Cv 0.10c 0.05c Other 20.3 6.80 27.0
Sc 0.44b 0.22a Saturated 25.3 60.3 24.1
ConsC 0.90a 0.14b Unsaturated 54.3 32.9 48.9

Results are the average of 23 replications. Results are the average of 23 replications.
Means followed by the same letter in the column did not differ signicantly from each Means followed by the same letter in a line, for the main fatty acids, did not differ
other, ScottKnott test, p b 0.05; F test, p b 0.05. signicantly from each other, ScottKnott test, p b 0.05.
 L. Gouveia et al. / Algal Research 16 (2016) 167176
for C. vulgaris [37,39], however Gouveia and Oliveira [40] found 5% lipid
content for the same Cv strain. Sc also presented lower lipid values than
usually reported in the literature (1729%) [37,41,42]. On the other
hand, ConsC has the highest lipid content (about 13.6%).
The fatty acid prole was determined for the three microalgae and
the results are shown in Table 7. Although the content of unidentied
fatty acids was quite signicant, namely for Cv and ConsC, the obtained
results suggested that lipids are mainly composed by unsaturated fatty
acids for Cv and ConsC and mostly composed of saturated fatty acids
(60%) for Sc. Palmitic acid (C16:0) was a signicant percentage of the
analysed algae (1837%). Among the unsaturated fatty acids, the
linolenic (C18:3) is the one with the highest content in Cv and ConsC,
however in Sc it is the oleic acid (C18:1) which is the highest. According
to EN 14214 [43], it is specied that for a biodiesel there is a limit of 12
and 1% for linolenic and polyunsaturated fatty acids (4 double bonds)
respectively and therefore the three algae do not meet specications.
For these microalgae to be used for biodiesel production they should
be associated with other oils. Nevertheless, due to their low content of
oils other biomass applications were depicted, such as BioH2 production
through dark fermentation [44], with a BioH2 yield that is even better
than when synthetic medium was used for cultivation. The potential
use of the produced biomass with wastewater as a biofertilizer was
also tested in Lactuca sativa (lettuce) seeds [45].
The protein values obtained for Cv was 56.4% which is much higher
than the ones usually reported in the literature (22% and 42% by Arbib
Fig. 4. Boundaries considered for cost analysis.
173
et al. [37] and He et al. [39]. respectively). Moreover, in the case of Sc,
the protein value was 32.7%. which is also higher than the ones reported
by Batista et al. [42] (20.4%), Arbib et al. [37] (16%) and Martinez et al.
[46](11.8%), although, Ruiz et al. [41] reported values between 19 and
37%. ConsC also presented a high value for protein (51.6%).
The carbohydrate content values in the microalgae studied were
27.7%, 20.6% and 11.7% for Cv, ConsC and Sc, respectively. These values
are within the range found by Batista et al. [47] for C. vulgaris (20%),
but higher than the ones found by He et al. [39] (14%). On the other
hand, Sc presented a sugar content lower than the one found by Miran-
da et al. [48] (32%) and Batista et al. [42] (30%), with both studies using
Bristol as a culture medium.
Cv had a total pigment content of 1.2%, similar to the one obtained by
Batista et al. [47], similar to Sc (1.2%) and ConsC had total pigment con-
tent of 0.9%. However, Hodaifa et al. [49] found a total pigment content
of 0.34% for Sc.
3.5. Cost evaluation
The cost analysis will fall on the PBR prototype (150 L) to identify
bottlenecks and recommendations for the scale-up and integration in
a WWTP.
The alternative microalgae production in the PBR was compared
with conventional means in terms of costs per 1 kg of dry biomass pro-
duced. Four pathways were considered for microalgae cultivation. In
174 L. Gouveia et al. / Algal Research 16 (2016) 167176

Path #1 the cultivation was performed in open ponds with sunlight, Table 9
fresh water and synthetic nutrients. In Path #2 the cultivation was Alternative production of 1 kg of Scenedesmus obliquus (Sc) by Path #3 and Path #4.

made in closed photobioreactors with articial light, fresh water and Process Input Specic Cost Path #3 Path #4
nutrients. In Paths #3 and #4 the cultivation was made in a closed /kWha kWh kWh
photobioreactor with sunlight and wastewater. In Paths #1, #2 and #3
Culture Electricity 0.096 177.4 17.03 177.4 17.03
the microalgae harvesting was made with centrifugation and the
Harvesting (centrifuge) Electricity 0.096 25 2.4 6.7 0.64
dewatering with a conventional oven, while Path #4 considered the al- Dewatering (oven) Electricity 0.096 0.2 0.017 0.1 0.01
ternative harvesting and dewatering using electrocoagulation as a pre- a
EDP Eletricidade de Portugal.
concentration step before centrifugation and a solar drying, respective-
ly. Human resources, infrastructure and maintenance costs are out of
the scope of the analysis. Operational boundaries are depicted in Fig. 4.
The conventional Sc produced by an open pond with 4500 L volume because of the high price of the nutrients. The comparison of the alter-
(Path #1) was taken from [50]. This pathway allows obtaining 1 kg of native harvesting and dewatering to the conventional one (full capaci-
dry microalga in 12 days consuming a signicant amount of water and ty) represents a decrease of 70% of energy consumed in these steps.
nutrients (Table 8). In a lab-scale 5 L bioreactor (Path #2) with articial With wastewater (Paths #3 and #4) huge costs are for electricity for
lighting 1 kg of the same microalga consumed even more water and nu- air blowing and pumping, yet the costs are still much lower than the
trients, with extremely low productivity levels (Table 8). The conven- price of nutrients.
tional Sc produced by the photobioreactor (Path #2) was taken from Other studies consider labour, depreciation costs, consumables, util-
[50]. ities and others. Nutrients (fertilizers) are considered in the category of
The growth of Sc by using wastewater in a 150 L bioreactor is able to raw material costs and water and power in the category of utilities costs
produce 1 kg of dry microalgae in 14 days with neither the addition of [52,53]. A target value of $0.5/kg is being widely agreed as the upper
fresh water nor nutrient resources (Table 9) (Paths #3 and #4). The av- limit [52].
erage productivity of 0.22 g/Ld was considered for the cost evaluation The pilot PBR is still far away from this target. If we look at the cost of
of Sc production with wastewater. However, the harvesting and treating 1 m3 of water, it would be roughly 95/m3 (air blower and
dewatering processes were different for both pathways. Centrifugation pump working for 14 days) which is pretty high compared with typical
and the electric oven were considered for Path #3 (Table 9), while values found on WWTP (0.100.2/m3) [54]. This is an indication that
electrocoagulation plus centrifugation and solar fan were considered electricity needs to be carefully sized for the 1500 L PBR and/or a
for Path #4 (Table 9). more efcient reactor and microalgae species with higher biomass pro-
The commercial value of Sc as of 2013 was 33/kg (http://www. ductivity must be used. Table 11 shows the cost for microalgae produc-
allma.com/pt). Table 10 shows the cost of production of 1 kg of dry tion found elsewhere.
microalga for our analysis. Typically these costs are within the range of 630% of the overall
Other studies estimate a microalgal biomass cost of $90$130/kg on costs. Wastewater use seems to be really effective in reducing
two vertical bioreactor designs with nutrient, water and electricity con- microalgae production costs. Moreover, Clarens et al. [55] showed that
sumption [51]. the use of wastewater reduced 50% of energy use and GHG emissions as-
It is quite interesting to observe that cultivation absorbs the majority sociated with chemical fertilizer production which was corroborated by
of costs even when conventional harvesting was used (Paths #1 and #2) Razon and Tan [56].
Regarding the scale-up from the 150 L to 1500 L some guidelines
may be useful towards a less energy consuming design: optimization
of aeration versus minimizing air blower and pump's energy consump-
Table 8 tion. The aeration used was 1 v/v/min and is relatable with the super-
Conventional production of 1 kg of Scenedesmus obliquus (Sc) by Path #1 and Path #2. cial gas velocity, based on the entire cross-sectional area of the reactor
Process Input Specic cost Path #1 Path #2 tube. The productivity was 0.22 g/Ld. This means an actual specic
Nutrient (kg) /gc g g
power (W = power needed for culture circulation and gas exchange
per m3 reactor working volume) as high as 20,600 W/m3 that trans-
NaNO3 0.10 239.36 23.19 1000 96.90
lates into an energy consumption of 1161 MJ/kg. For the 1 v/v/min
K2HPO4 0.09 71.81 6.35 300 26.52
MgSO47H2O 0.06 71.81 6.43 300 26.88 and 150 L reactor the air ow rate would be 150 L/min. The air compres-
CaCl2H2O 0.04 31.60 1.63 132 6.80 sor power could be as low as 150 W [57]. In fact, Alexander Burns [57],
NaCl 0.03 23.94 10.96 100 45.80 found an optimal specic power input in the range of 330 to
Fe-EDTA 0.11 57.45 8.67 120 18.12 360 Wm3 for a vertical bioreactor to get a maximum productivity of
KH2PO4 0.11 167.55 19.24 700 80.36
H3BO3 0.05 2.74 0.30 1144 126.98
2 g/Ld (aeration rate of 0.35 v/v/min) resulting in 17.6 to 19.1 mg/kJ
Culture MnCl24H2O 0.15 1.94 2.27 812 950.04 biomass (equivalent to 56.852.4 MJ energy consumed to produce
ZnSO47H2O 0.24 0.21 0.02 88 9.59 1 kg biomass).
CuSO4 0.29 0.05 0.003 20 1.16 For the 1500 L bioreactor some design considerations must therefore
Na2MoO42H2O 0.32 0.06 0.002 24 0.86
be undertaken in order to reduce drastically the energy consumption of
CoCl27H2O 1.17 0.09 0.008 36 3.21
CO2 (m3) 0.13/m3d NA NA NA NA
Water (m3) 1.64 /m3a 1.0 1.64 1.7 2.79
Electricity 0.4
0.096 /kWhb 0.039 237.17 22.8
(kWh) kWh Table 10
Harvesting Electricity b 25 25 Production cost inventory in /kg.
0.096 /kWh 2.4 2.4
(centrifuge) (kWh) kWh kWh
Stage Path #1 Path #2 Path #3 Path #4
Dewatering Electricity 0.2 0.2
0.096 /kWhb 0.017 0.017
(oven) (kWh) kWh kWh Culturenutrients 79.09 1161.02 0.00 0.00
Culturewater 1.64 2.79 0.00 0.00
NA not available.
a Cultureelectricity 0.04 22.77 30.96 30.96
EPAL Empresa Portuguesa das guas Livres s.a. Portugal.
b Harvesting 2.40 2.40 2.40 0.64
EDP Eletricidade de Portugal.
c Drying 0.02 0.02 0.02 0.01
http://www.sigmaaldrich.com/catalog/AdvancedSearchPage.do
d Total 83.19 1188.99 33.38 31.61
Air liquide https://www.airliquide.com/.
 L. Gouveia et al. / Algal Research 16 (2016) 167176
Table 11
Cost of microalgae production from literature.
Microalgae Reactor Raw materials
Fertilizers
Haematococcus pluvialis Tubular raceway pond 7.32
Scenedesmus almeriensisa Tubular 0.78
a
Can go down to 12/kg if technology is simplied and scaled-up to a production capacity of 200 t/year [53].
the system (and operational cost) and take advantage of the nutrient
and fresh water avoidance costs. The air compressor power could be es-
timated by the following equation:
Pac W 1:0346  mair L= min:
The desired aeration rate (aer in v/v/min or L/L/min) could be related
to the air ow rate (mair) and bioreactor capacity (VBio) by:
mair aer L=L= min  VBio L:
The pump power could be chosen by estimating the friction losses
throughout the pipes, using uid mechanic equations: Reynolds num-
ber, relative roughness, Moody's diagram, pump efciency and energy
balance equation [58].
So assuming an aeration rate of 0.35 v/v/day the specic power of
360 W/m3 and 0.22 g/Ld, means 90 MJ of energy consumed per 1 kg
biomass which will translate in 2.4/kg or 1.6/m3 treated water.
4. Conclusion
This ongoing project intends to treat urban wastewater using a
photobioreactor inoculated with microalgae.
The performance of the PBR was quite interesting, mainly for phos-
phorous and total nitrogen removal. The resulting clean water is in ac-
cordance to the environmental legislation (Directive 98/15/CE) and
therefore the water could be used without restrictions or discharge in
water ow. These results prove that wastewater treatment by
microalgae is technically possible.
Among the three algae used, the results were very similar regarding
nutrient removal rates, however Consortium C gave the best results in
terms of removal and biomass productivity and quality (for further
uses) but took longer. This is an expected result, due to the fact that
this consortium of algae was isolated from the efuent. Nevertheless,
the Scenedesmus obliquus gave almost the same results in terms of nutri-
ent removal in less time. Considering the microalgae biomass composi-
tion, which is quite interesting for biofuel production, in particular for
ethanol/biohydrogen/biogas the best biomass were Cv and Consortium
C.
Combining wastewater treatment with biomass productivity and
quality, Consortium C is probably the best option (if the edaphoclimatic
condition was favourable).
Moreover, the exploration of new microalgae strains that might be
able to grow naturally in the wastewaters will bring the opportunity
to harness the potential of local biodiversity.
Regarding costs the use of microalgae to produce clean water must
be carefully analysed to achieve the same levels of cost as conventional
wastewater plants (0.2/m3). This implies the reduction of the electrical
energy from the grid needed for the air compressor and pump systems.
This electrical energy needs are still high (323 kWh/kg) and need a dras-
tic reduction by a correct aeration rate determination and adequate air
compressor and pump dimensioning to achieve the production cost tar-
get value of $0.5/kg.
175
Utilities Total Production cost (/kg)
CO2 Water Power
5.25 4.14 16.52 33.22 18.00
1.92 0.06 3.14 5.90 89.00
Acknowledgements
This study was supported by the project WW-SIP From Urban
Wastewater Treatment Plant to Self-Sustainable Integrated Platform
for Wastewater Renement (LIFE10 ENV/IT/000308).
The authors would like to acknowledge the Fundao para a Cincia
e Tecnologia for the nancial support of Ana F. Ferreira through Post-
Doc grant no. SFRH/BPD/95098/2013 and support for Carla M. Silva to
the 2012 FCT researcher competition (IF/00181/2012). The authors
would like to acknowledge the project IDL-UID/GEO/50019/2013.
The authors would like to acknowledge Dr. Lina Baeta-Hall for the
strain isolation, Dr. Ana Cristina Oliveira for the fatty acid analysis,
Eng. David Loureiro for the solar oven, and Cu Penedo, Graa Gomes
and Natrcia Santos for microalgae culture maintenance and laboratory
assistance.
References
[1] J.C. Pasqualino, M. Meneses, F. Castells, Life cycle assessment of urban wastewater
reclamation and reuse alternatives, J. Ind. Ecol. 15 (2011) 4963, http://dx.doi.org/
10.1111/j.1530-9290.2010.00293.x.
[2] W. Mo, Q. Zhang, Can municipal wastewater treatment systems be carbon neutral?
J. Environ. Manag. 112 (2012) 360367, http://dx.doi.org/10.1016/j.jenvman.2012.
08.014.
[3] M. Meneses, J.C. Pasqualino, F. Castells, Environmental assessment of urban waste-
water reuse: treatment alternatives and applications, Chemosphere 81 (2010)
266272, http://dx.doi.org/10.1016/j.chemosphere.2010.05.053.
[4] I. Muoz, L. Mil-i-Canals, A.R. Fernndez-Alba, Life cycle assessment of water sup-
ply plans in Mediterranean Spain, J. Ind. Ecol. 14 (2010) 902918, http://dx.doi.org/
10.1111/j.1530-9290.2010.00271.x.
[5] G. Houillon, O. Jolliet, Life cycle assessment of processes for the treatment of waste-
water urban sludge: energy and global warming analysis, J. Clean. Prod. 13 (2005)
287299, http://dx.doi.org/10.1016/j.jclepro.2004.02.022.
[6] A. Hospido, T. Moreira, M. Martn, M. Rigola, G. Feijoo, Environmental evaluation of
different treatment processes for sludge from urban wastewater treatments: anaer-
obic digestion versus thermal processes(10 pp) Int. J. Life Cycle Assess. 10 (2005)
336345, http://dx.doi.org/10.1065/lca2005.05.210.
[7] J. Nouri, K. Nadda, R. Nabizadeh, M. Jafarinia, Energy recovery from wastewater
treatment plant, Pak. J. Biol. Sci. 9 (2006) 36, http://dx.doi.org/10.3923/pjbs.2006.
3.6.
[8] W.J. Oswald, My sixty years in applied algology, J. Appl. Phycol. 15 (2003) 99106,
http://dx.doi.org/10.1023/A:1023871903434.
[9] F.B. Green, L.S. Bernstone, T.J. Lundquist, W.J. Oswald, Advanced integrated waste-
water pond systems for nitrogen removal, Water Sci. Technol. 33 (1996) 207217,
http://dx.doi.org/10.1016/0273-1223(96)00356-3.
[10] M.-O.P. Fortier, B.S.M. Sturm, Geographic analysis of the feasibility of collocating
algal biomass production with wastewater treatment plants, Environ. Sci. Technol.
46 (2012) 1142611434, http://dx.doi.org/10.1021/es302127f.
[11] E. Molina Grima, E.H. Belarbi, F.G. Acin Fernndez, a. Robles Medina, Y. Chisti, Recovery
of microalgal biomass and metabolites: process options and economics, Biotechnol.
Adv. 20 (2003) 491515, http://dx.doi.org/10.1016/S07349750(02)000502.
[12] C.T. Matos, M. Santos, B.P. Nobre, L. Gouveia, Nannochloropsis sp. biomass recovery
by electro-coagulation for biodiesel and pigment production, Bioresour. Technol.
134 (2013) 219226, http://dx.doi.org/10.1016/j.biortech.2013.02.034.
[13] E. Poelman, N. De Pauw, B. Jeurissen, Potential of electrolytic occulation for recov-
ery of micro-algae, Resour. Conserv. Recycl. 19 (1997) 110, http://dx.doi.org/10.
1016/S0921-3449(96)011561.
[14] C.G. Alfafara, K. Nakano, N. Nomura, T. Igarashi, M. Matsumura, Operating and scale-
up factors for the electrolytic removal of algae from eutrophied lakewater, J. Chem.
Technol. Biotechnol. 77 (2002) 871876, http://dx.doi.org/10.1002/jctb.649.
[15] G. Azarian, A. Mesdaghinia, F. Vaezi, R. Nabizadeh, D. Nematollahi, Algae Removal by
Electro-coagulation Process, Application for Treatment of the Efuent from an In-
dustrial Wastewater Treatment Plant, 362007 5764.
[16] S. Gao, J. Yang, J. Tian, F. Ma, G. Tu, M. Du, Electro-coagulationotation process for
algae removal, J. Hazard. Mater. 177 (2010) 336343, http://dx.doi.org/10.1016/j.
jhazmat.2009.12.037.
[17] S. Gao, M. Du, J. Tian, J. Yang, J. Yang, F. Ma, et al., Effects of chloride ions on electro-
coagulationotation process with aluminum electrodes for algae removal, J. Haz-
ard. Mater. 182 (2010) 827834, http://dx.doi.org/10.1016/j.jhazmat.2010.06.114.
176 L. Gouveia et al. / Algal Research 16 (2016) 167176
[18] D. Vandamme, S.C.V. Pontes, K. Goiris, I. Foubert, L.J.J. Pinoy, K. Muylaert, Evaluation
of electro-coagulationocculation for harvesting marine and freshwater
microalgae, Biotechnol. Bioeng. 108 (2011) 23202329, http://dx.doi.org/10.1002/
bit.23199.
[19] N. Uduman, V. Bourniquel, M.K. Danquah, A.F.a. Hoadley, A parametric study of
electrocoagulation as a recovery process of marine microalgae for biodiesel produc-
tion, Chem. Eng. J. 174 (2011) 249257, http://dx.doi.org/10.1016/j.cej.2011.09.012.
[20] National Alliance for Advanced Biofuels and Bio-products, Final Report Synopsis,
http://energy.gov/eere/bioenergy/downloads/national-alliance-advanced-biofuels-
and-bioproducts-synopsis-naabb-nal2014.
[21] R. Pacheco, A.F. Ferreira, T. Pinto, B.P. Nobre, D. Loureiro, P. Moura, et al., The produc-
tion of pigments & hydrogen through a Spirogyra sp. biorenery, Energy Convers.
Manag. 89 (2015) 789797, http://dx.doi.org/10.1016/j.enconman.2014.10.040.
[22] R. Voskens, F. Zegers, Solar Drying in Europe, 2005.
[23] A. Nogueira, E. Atade, M. Joo, S. Enoch, D. Loureiro, A. Cruz, Simulation and Control
Strategies for an Energetically Efcient Wood Drying Process, 2005 244251.
[24] L. Brennan, P. Owende, Biofuels from microalgaea review of technologies for pro-
duction, processing, and extractions of biofuels and co-products, Renew. Sust. Energ.
Rev. 14 (2010) 557577, http://dx.doi.org/10.1016/j.rser.2009.10.009.
[25] IPMA - Instituto Portugus do Mar e da Atmosfera, Https://www.ipma.pt/pt/
otempo/prev.descritiva/inde2014.
[26] R.C. Starr, J.A. Zeikus, UTEXthe culture collection of algae at the University of Texas
at Austin 1993 list of cultures, J. Phycol. 29 (1993) 1106, http://dx.doi.org/10.1111/
j.0022-3646.1993.00001.x.
[27] EN ISO 5509:2000, Animal and Vegetable Fats and OilsPreparation of Methyl Es-
ters of Fatty Acids, 2000.
[28] B.P. Nobre, F. Villalobos, B.E. Barragn, A.C. Oliveira, A.P. Batista, P.A.S.S. Marques,
et al., A biorenery from Nannochloropsis sp. microalgaextraction of oils and pig-
ments. Production of biohydrogen from the leftover biomass, Bioresour. Technol.
135 (2013) 128136, http://dx.doi.org/10.1016/j.biortech.2012.11.084.
[29] EN 14103:2003, Fat and Oil Derivatives Fatty Acid Methyl Esters (FAME) Deter-
mination of Ester and Linolenic Acid Methyl Ester Contents, 2003.
[30] C. Hoebler, J. Barry, Rapid acid hydrolysis of plant cell wall polysaccharides and sim-
plied quantitative determination of their neutral monosaccharides by gasliquid
chromatography, J. Agric. (1989) 360367, http://dx.doi.org/10.1021/jf00086a020.
[31] 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) 350356,
http://dx.doi.org/10.1021/ac60111a017.
[32] W. Horwitz, G.W. Latimer, A.O.A.C., Ofcial Methods of Analysis, 18th ed., 2005.
[33] D.B. Jones, Factors for Converting Percentages of Nitrogen in Foods and Feeds into
Percentages of Protein, 1931, http://dx.doi.org/10.1163/_q3_SIM_00374.
[34] L. Gouveia, V. Veloso, A. Reis, H. Fernandes, J. Novais, J. Empis, Evolution of pigment
composition in Chlorella vulgaris, Bioresour. Technol. 57 (1996) 157163, http://dx.
doi.org/10.1016/0960-8524(96)00058-2.
[35] L. Gouveia, J. Empis, Relative stabilities of microalgal carotenoids in microalgal extracts,
biomass and sh feed: effect of storage conditions, Innovative Food Sci. Emerg. Technol.
4 (2003) 227233, http://dx.doi.org/10.1016/S1466-8564(03)00002-X.
[36] D.F. Ferreira, SISVAR: a computer statistical analysis system, Cienc. E Agrotecnologia.
35 (2011) 10391042, http://dx.doi.org/10.1590/S1413-70542011000600001.
[37] Z. Arbib, J. Ruiz, P. lvarez-Daz, C. Garrido-Prez, J.a. Perales, Capability of different
microalgae species for phytoremediation processes: wastewater tertiary treatment,
CO2 bio-xation and low cost biofuels production, Water Res. 49 (2014) 465474,
http://dx.doi.org/10.1016/j.watres.2013.10.036.
[38] C.M. Silva, A.F. Ferreira, A.P. Dias, M. Costa, A comparison between microalgae virtu-
al biorenery arrangements for bio-oil production based on lab-scale results, J.
Clean. Prod. (2015), http://dx.doi.org/10.1016/j.jclepro.2015.09.053.
[39] P.J. He, B. Mao, C.M. Shen, L.M. Shao, D.J. Lee, J.S. Chang, Cultivation of Chlorella
vulgaris on wastewater containing high levels of ammonia for biodiesel production,
Bioresour. Technol. 129C (2012) 177181, http://dx.doi.org/10.1016/j.biortech.
2012.10.162.
[40] L. Gouveia, A.C. Oliveira, Microalgae as a raw material for biofuels production, J. Ind.
Microbiol. Biotechnol. 36 (2009) 269274, http://dx.doi.org/10.1007/s10295-008-
0495-6.
[41] J. Ruiz, P.D. lvarez-Daz, Z. Arbib, C. Garrido-Prez, J. Barragn, J.a. Perales, Perfor-
mance of a at panel reactor in the continuous culture of microalgae in urban
wastewater: prediction from a batch experiment, Bioresour. Technol. 127 (2013)
456463, http://dx.doi.org/10.1016/j.biortech.2012.09.103.
[42] A.P. Batista, P. Moura, P.A.S.S. Marques, J. Ortigueira, L. Alves, L. Gouveia,
Scenedesmus obliquus as feedstock for biohydrogen production by Enterobacter
aerogenes and Clostridium butyricum, Fuel 117 (2014) 537543, http://dx.doi.org/
10.1016/j.fuel.2013.09.077.
[43] EN 14214, UNE-EN 14214:2013 automotive fuels. Fatty acid methyl esters (FAME)
for diesel engines, Requir. test. methods 22 (2013) (doi:ISBN 978 0 580 70781 0).
[44] A.P. Batista, L. Ambrosano, S. Graa, C. Sousa, P.A.S.S. Marques, B. Ribeiro, et al., Com-
bining urban wastewater treatment with biohydrogen production an integrated
microalgae-based approach, Bioresour. Technol. 184 (2015) 230235, http://dx.
doi.org/10.1016/j.biortech.2014.10.064.
[45] L. Ambrosano, Microalgae Chemical Composition and the Effect of the Extracts on
Seed Germination, University of Lavras, Brasil, 2015.
[46] M. Martnez, S. Sanchez, J.M. Jimnez, E. Yous, L. Munoz, Nitrogen and phosphorus
removal from urban wastewater by the microalga Scenedesmus obliquus, Bioresour.
Technol. 73 (2000) 263272, http://dx.doi.org/10.1016/S0960-8524(99)00121-2.
[47] A.P. Batista, L. Gouveia, N.M. Bandarra, J.M. Franco, A. Raymundo, Comparison of
microalgal biomass proles as novel functional ingredient for food products, Algal
Res. 2 (2013) 164173, http://dx.doi.org/10.1016/j.algal.2013.01.004.
[48] J.R. Miranda, P.C. Passarinho, L. Gouveia, Pre-treatment optimization of Scenedesmus
obliquus microalga for bioethanol production, Bioresour. Technol. 104 (2012)
342348, http://dx.doi.org/10.1016/j.biortech.2011.10.059.
[49] G. Hodaifa, M.E. Martnez, S. Snchez, Inuence of temperature on growth of
Scenedesmus obliquus in diluted olive mill wastewater as culture medium, Eng.
Life Sci. 10 (2010) 257264, http://dx.doi.org/10.1002/elsc.201000005.
[50] A.F. Ferreira, J. Ortigueira, L. Alves, L. Gouveia, P. Moura, C. Silva, Biohydrogen pro-
duction from microalgal biomass: energy requirement, CO2 emissions and scale-
up scenarios, Bioresour. Technol. 144 (2013) 156164, http://dx.doi.org/10.1016/j.
biortech.2013.06.079.
[51] S. Monkonsit, S. Powtongsook, P. Pavasant, Comparison between airlift
photobioreactor and bubble column for Skeletonema costatum cultivation, Engl. J.
15 (2011) 5364, http://dx.doi.org/10.4186/ej.2011.15.4.53.
[52] F.G. Acin, J.M. Fernndez, E. Molina-Grima, Economics of Microalgae Biomass Pro-
duction, Biofuels from Algae, 2013 313325, http://dx.doi.org/10.1016/B978-0-
444-59558-4.00014-0.
[53] F.G. Acin, J.M. Fernndez, J.J. Magn, E. Molina, Production cost of a real microalgae
production plant and strategies to reduce it, Biotechnol. Adv. 30 (2012) 13441353,
http://dx.doi.org/10.1016/j.biotechadv.2012.02.005.
[54] S. Cashman, A. Gaglione, A. Mosley, E. Al, Environmental and Cost Life Cycle Assess-
ment of Disinfection Options for Municipal Wastewater Treatment, 2014.
[55] A.F. Clarens, E.P. Resurreccion, M. a White, L.M. Colosi, Environmental life cycle com-
parison of algae to other bioenergy feedstocks, Environ. Sci. Technol. 44 (2010)
18131819, http://dx.doi.org/10.1021/es902838n.
[56] L.F. Razon, R.R. Tan, Net energy analysis of the production of biodiesel and biogas
from the microalgae: Haematococcus pluvialis and Nannochloropsis, Appl. Energy
88 (2011) 35073514, http://dx.doi.org/10.1016/j.apenergy.2010.12.052.
[57] A. Burns, Photobioreactor Design for Improved Energy Efciency of Microalgae Pro-
duction, Faculty of California Polytechnic State University, San Luis Obispo, 2004.
[58] C. Geankoplis, Transport Processes and Separation Process Principles (Includes Unit
Operations), fourth ed. Prentice Hall Press, Upper Saddle River, NJ, USA, 2003.