Journal of Cleaner Production 133 (2016) 348e357

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Journal of Cleaner Production

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Wastewater polishing by consortia of Chlorella vulgaris and activated

sludge native bacteria
~ es*

C.M. Pires, Manuel Simo
Ana L. Gonçalves, Jose
LEPABE, Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal

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

Article history: The increase of human population and anthropogenic activities has resulted in an excessive discharge of
Received 23 November 2015 wastes into water bodies, contributing to nutrients enrichment and consequent degradation of aquatic
Received in revised form environments and freshwater resources. The need for cost-effective systems able to reduce nutrients
11 May 2016
concentration in wastewaters is therefore a major concern. Microalgal-bacterial consortia can be effec-
Accepted 18 May 2016
tively applied in wastewater polishing, since co-cultivation of these microorganisms can promote mutual
Available online 26 May 2016
growth and nutrients removal from wastewaters. In this study, dual-species cultures of the microalga
Chlorella vulgaris and a bacterium isolated from a municipal wastewater treatment plant (Enterobacter
Keywords:
Biomass production
asburiae, Klebsiella sp. or Raoultella ornithinolytica) were performed. Studying these consortia aimed the
Microalgal-bacterial consortium evaluation of their potential in nutrients (nitrogen, phosphorus and chemical oxygen demand) removal
Nutrients enrichment from a synthetic medium that mimics a secondary-treated effluent (nitrogen, phosphorus and chemical
Wastewater polishing oxygen demand loads of about 45 mg N L
1, 10 mg P L
1 and 300 mg O2 L
1, respectively). The results
have shown that the consortia containing E. asburiae and R. ornithinolytica have significantly improved
C. vulgaris growth: when in single cultures maximum cell concentration achieved by C. vulgaris was
(8.27 ± 0.79)  106 cells per mL, whereas in these consortia maximum cell concentrations achieved by
C. vulgaris were (19.8 ± 2.0)  106 and (14.4 ± 2.3)  106 cells per mL, respectively. The three studied
consortia have also contributed to higher nutrients removal, since the time required to achieve the limits
in discharged effluents (established by European Union legislation) was reduced to at least half of the
value determined for the single C. vulgaris culture. Due to the effectiveness demonstrated by the studied
consortia, these microalgal-bacterial systems should be considered as a viable approach for application in
the tertiary treatment step of wastewater treatment processes.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction effective treatment solutions able to reduce nitrogen and phos-

The rapid population increase has conducted the world to a non-
uniform distribution of water resources: (i) freshwater resources
are becoming limited; and (ii) the increase of anthropogenic ac-
tivities, such as agricultural practices, urbanization and industrial-
ization, result in an excessive dumping of wastes into aquatic
bodies, increasing nutrients (mainly nitrogen and phosphorus) in-
puts (Aslan and Kapdan, 2006; Rawat et al., 2011; Renuka et al.,
2013). Nutrients enrichment or eutrophication results in the
development of algal blooms, spread of aquatic plants, oxygen
depletion, loss of key species and degradation of freshwater eco-
systems (Renuka et al., 2013; Ruiz et al., 2013). For these reasons,
* Corresponding author. Tel.: þ351 22 508 1654; fax: þ351 22 508 1449.
~es).
E-mail address: mvs@fe.up.pt (M. Simo
phorus concentrations in wastewaters before discharging in natural
bodies are required. Accordingly, European Union (EU) Directives
1991/271/EEC and 1998/15/EC have defined limits for nutrients
concentrations in discharged effluents, as well as minimum per-
centage load reductions (Aslan and Kapdan, 2006; Renuka et al.,
2013). In the secondary treatment step of wastewater treatment
processes, ammonia and organic matter are oxidized by hetero-
trophic bacteria, also known as activated sludge, resulting in an
effluent rich in nitrates, nitrites and phosphates. These nutrients
can then be removed in the tertiary treatment step, in order to
promote wastewater polishing. The most commonly used methods
for wastewater polishing include biological processes, such as
anaerobic digestion followed by nitrification and denitrification
(Fulazzaky et al., 2015; Piao et al., 2015). However, several nitrifi-
cation and denitrification cycles are required to achieve the
nutrient levels accepted by EU legislation. Additionally, these

http://dx.doi.org/10.1016/j.jclepro.2016.05.109
0959-6526/© 2016 Elsevier Ltd. All rights reserved.
 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357
methods require several tanks, internal recycles of activated sludge
and long hydraulic residence times, resulting in an overall increase
of process costs, complexity and energy input (Foess et al., 1998;
Jeyanayagam, 2005; Singh and Thomas, 2012). Alternatively, ni-
trogen and phosphorus removal may be achieved by chemical
methods, such as precipitation using aluminium and iron salts.
However, these methods are costly and produce large amounts of
sludge contaminated with chemical compounds (Barnard, 1975;
Wang et al., 2006; Zang et al., 2015). To overcome these draw-
backs, the development of alternative methods for wastewater
polishing requiring lower energy inputs and producing smaller
amounts of waste at reduced costs are required.
Cultivation of microalgae, a broad category of photosynthetic
microorganisms comprising eukaryotic microalgae and prokaryotic
cyanobacteria, in wastewaters appears as a viable alternative for the
reduction of nitrogen and phosphorus levels, since these microor-
ganisms require high amounts of nitrogen and phosphorus for their
growth (Cuellar-Bermudez et al., 2015; Gupta et al., 2015; Monari
et al., 2016). The mechanisms involved in nutrients removal by
microalgae include: for nitrogen, the assimilation of both nitrate
and ammonia and the stripping of ammonia due to the increase in
pH during microalgal growth; and for phosphorus, the bio-
assimilation, adsorption and chemical precipitation due to pH
values above 8 and high dissolved oxygen concentrations (Aslan
and Kapdan, 2006; Cai et al., 2013; Wang et al., 2014). Recently,
several authors have demonstrated the potential of microalgal-
bacterial associations as an alternative to the secondary treat-
ment step of wastewater treatment (Gonza
lez-Ferna

ndez et al.,
2011; Medina and Neis, 2007; Su et al., 2011). In these associa-
tions, microalgae and bacteria interact cooperatively. Through
photosynthesis, microalgae supply heterotrophic bacteria with
organic compounds released during algal growth and with O2 that
is required for the oxidation of the organic matter (Bordel et al.,
2009; De Godos et al., 2009). Microalgae can also serve as a
habitat for bacteria, protecting them from adverse environmental
conditions (Unnithan et al., 2014). Alternatively, bacteria supply
microalgae with the CO2 required for photosynthesis and with
organic growth factors and vitamins that enhance microalgal
growth (Bordel et al., 2009; De Godos et al., 2009; Unnithan et al.,
2014). These cooperative interactions present several advantages
(Aslan and Kapdan, 2006; Manninen et al., in press; Rawat et al.,
2011): (i) nitrogen and phosphorus assimilated by microalgae can
be recycled by the production of fertilizers from microalgal
biomass; (ii) the resulting biomass can also be used for the pro-
duction of bioenergy; and (iii) an oxygenated effluent is discharged
into the water bodies. Selection of the microorganisms to integrate
these consortia is an important issue to address when considering
the use of algal-bacterial consortia for wastewater treatment. The
selected microorganisms may be able to grow in wastewaters and
in similar environmental conditions as those found in wastewater
treatment plants.
Although microalgal-bacterial consortia have already been
applied to replace the secondary treatment step, this study pro-
poses an alternative wastewater polishing method based on these
cooperative interactions for application in already existing waste-
water treatment plants lacking a tertiary treatment phase.
Accordingly, this work aimed at (i) understanding the behaviour of
Chlorella vulgaris and bacteria in dual-species consortia and to infer
possible synergistic and/or competitive mechanisms; and (ii) to
evaluate nutrients (nitrogen e N, phosphorus e P and chemical
oxygen demand e COD) removal performance of the dual
microalgal-bacterial consortia compared to the one of single spe-
cies, in order to assess the feasibility of these consortia to treat
secondary-treated wastewater. The microalga selected for the
development of these consortia was C. vulgaris, a microorganism
349
widely applied in wastewater treatment processes, due to its high
ability for nutrients removal (Wang et al., 2010). This microalga was
co-cultured with different bacteria isolated and identified from the
activated sludge tank of a local municipal wastewater treatment
plant (MWTP). Activated sludge native bacteria were selected to
constitute these consortia because these microorganisms were
already adapted to the environmental conditions typically found in
a MWTP.
2. Materials and methods
2.1. Microorganisms and culture medium
The microalga C. vulgaris CCAP 211/11B was obtained from
Culture Collection of Algae and Protozoa (United Kingdom). Stock
solutions of this microalga were prepared in a synthetic secondary
effluent (Gebara, 1999), with the following composition (per litre):
300 mg C6H12O6, 250 mg NaNO3, 10 mg MgSO4$7H2O, 45 mg
KH2PO4, 1 mg MnSO4$H2O, 0.46 mg CaCl2$2H2O, 0.05 mg
FeCl3$6H2O and 0.06 mg Na2EDTA$2H2O (pH ¼ 6.39 ± 0.46). Cul-
ture medium was sterilized by autoclaving at 121  C for 15 min. The
cells were incubated in 500 mL flasks (VWR, Portugal) at room
temperature (22.3 ± 0.7  C), under continuous fluorescent light
with an irradiance of 120 mE m
2 s
1 at the surface of the flasks.
Agitation was obtained by bubbling atmospheric air (filtered
through 0.22 mm cellulose acetate membranes, Orange Scientific,
Belgium) at the bottom of the flasks. After cultivation for one week,
cells were harvested through centrifugation at 2900 g for 15 min (in
an Eppendorf 5810 R centrifuge, Eppendorf, Germany) and appro-
priate dilutions in the above described medium were performed, to
obtain an inoculum concentration of approximately 1.0  106 cells
per mL.
From a sample collected from the secondary treatment step of
the MWTP located in Rabada (Santo Tirso, Aguas
de Portugal), in
May 2014, three microorganisms able to grow on the above
described synthetic wastewater have been isolated. These micro-
organisms were identified (STABVIDA, Portugal) through 16S rRNA
gene sequencing using four different primers: 27F, 518F, 800R and
1492R. Identification of the three isolates from the MWTP has
distinguished three rod-shaped Gram-negative bacteria belonging
to the family Enterobacteriaceae (phylum Proteobacteria): Enter-
obacter asburiae, Klebsiella sp. and Raoultella ornithinolytica,
formerly known as Klebsiella ornithinolytica. These microorganisms
are commonly found in water, soil, plants, sewage and others. After
the isolation and identification steps, these isolates were grown in
250 mL Erlenmeyer flasks using Luria Broth as culture medium.
Cells were incubated overnight at 30  C with orbital agitation
(150 rpm). After the incubation period, cells were harvested
through centrifugation at 2900 g for 15 min (in an Eppendorf 5810
R centrifuge, Eppendorf, Germany) and appropriate dilutions in the
above described synthetic medium were performed, to obtain an
inoculum concentration of approximately 1.0  108 colony forming
units (CFU) per mL.
2.2. Microalgal and bacterial growth in single cultures and in the
consortia
The study of C. vulgaris co-culture with the bacterial isolates has
resulted in three different consortia: C. vulgaris þ E. asburiae (CE
consortium), C. vulgaris þ Klebsiella sp. (CK consortium) and
C. vulgaris þ R. ornithinolytica (CR consortium). Initial cell concen-
trations of microalgae and bacteria were identical to those of the
single species cultures used as control: 1.0  106 cells per mL and
1.0  108 CFU per mL, respectively. Batch experiments were per-
formed for seven days in 500 mL flasks (VWR, Portugal) with a
350 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357
working volume of 400 mL. Temperature, light and aeration con-
ditions were the same as for C. vulgaris stock solutions preparation.
For each biological system, four independent experiments were
performed under aseptic conditions. Within the cultivation time,
temperature, pH and dissolved oxygen concentration were daily
monitored: pH was measured using a pH212 (Hanna Instruments,
Germany) microprocessor-based pH meter, whereas temperature
and dissolved oxygen were monitored using an Oxi 340i (WTW,
Germany) oxygen sensor.
2.3. Growth monitoring and kinetic growth parameters
Duplicate samples were collected at 2 h intervals for 8 h and
then at 24, 48, 72, 96, 120, 144 and 168 h. Cell concentration of
C. vulgaris, NM (cells per mL), was determined using a Neubauer
counting chamber (Marienfeld, Germany), under a Leica DM LB
(Leica Microsystems, Germany) microscope. On the other hand, cell
concentration of each isolate, NB (CFU per mL), was determined
through CFU enumeration. For this, the necessary dilutions were
prepared and plated on Plate Count Agar (PCA, VWR, Portugal)
using the motion drop method (Reed and Reed, 1948) and plates
were incubated at 30  C for 16 h. Additionally, duplicate samples
(40 mL) were collected in the first and last day of culturing to
determine biomass concentration in terms of ash free dry weight
(DW). In these determinations, the collected samples were inserted
in previously dried porcelain crucibles. The samples were allowed
to dry for 48 h at 105  C until a constant weight was obtained. The
dried biomass was then oxidized at 550  C for 2 h in a muffle and
re-weighed. The mass loss obtained after biomass oxidation
divided by the samples' volume corresponds to the ash free dry
weight biomass concentration, X (g DW L
1). Average biomass
productivities, P (g DW L
1 d
1), were calculated from the variation
in biomass concentration within the cultivation time, as shown in
Equation (1) (Feng et al., 2012; Jacob-Lopes et al., 2009):
Xf
Xi
P¼
tf
ti
where Xf and Xi correspond to biomass concentration (in g DW L
1)
at times tf and ti (in days), the end and beginning of cultivation time,
respectively.
2.4. Nutrients removal
Since EU legislation imposes limits for nutrients concentrations
in discharged effluents, as well as minimum percentage load re-
ductions (Directive 1991/271/EEC, 1991; Directive 1998/15/EC,
1998), nitrogen, phosphorus and COD levels were determined
within the cultivation time to evaluate the potential of the studied
cultures in nutrients removal. Taking into account the definition of
population equivalent (PE), the limits for effluent discharge are: (i)
15 mg N L
1 (10e100 thousand PE) or 10 mg N L
1 (more than 100
thousand PE) for total nitrogen with a minimum percentage of
reduction of 70e80%; (ii) 2 mg P L
1 (10e100 thousand PE) or
1 mg P L
1 (more than 100 thousand PE) for total phosphorus with
a minimum percentage of reduction of 80%; and (iii) 125 mg O2 L
1
for COD with a minimum percentage of reduction of 75%. Duplicate
samples were centrifuged at 2900 g for 15 min using an Eppendorf
5810 R centrifuge (Eppendorf, Germany) and supernatants were
analysed.
Nitrate concentration was determined through UV spectroscopy
at 220 nm using a T80 UV/VIS Spectrophotometer (PG Instruments,
UK), according to the method proposed by Collos et al. (1999).
Distilled water previously filtered through 0.22 mm cellulose ace-
tate membranes (Orange Scientific, Belgium) was used as blank.
The calibration curve was determined by preparing NaNO3 stan-
dards with concentrations ranging from 5 to 500 mg L
1 and sub-
mitting them to the same procedure as the analysed samples.
Inorganic phosphate quantification was performed by
measuring absorbance at 820 nm of a phosphomolybdate complex
formed by reaction of inorganic phosphate with ammonium
molybdate in a Synergy™ HT 96-well microplate reader (Biotek
Instruments, Inc., USA), as proposed by Lee et al. (2009). The blank
was measured by repeating this procedure using distilled water. To
determine the calibration curve, standards of KH2PO4 with con-
centrations ranging from 1 to 60 mg L
1 were submitted to the
same procedure as the analysed samples.
Measurements of soluble COD were performed using the closed
reflux titrimetric method according to Standard Methods (APHA,
1999). The samples were refluxed in a strong acidic solution with
a known excess of potassium dichromate (K2Cr2O7). After digestion,
the amount of consumed K2Cr2O7 was determined by titration of
the unreduced K2Cr2O7 with ferrous ammonium sulphate and the
organic matter was calculated in terms of oxygen equivalent.
Values of nutrients concentrations within the cultivation time
were used to determine nutrients removal efficiencies (R, in %) and
uptake rates (k, in d
1).
Nutrients removal efficiencies were determined according to
Equation (2):
Si
Sf
%R ¼ $100 (2)
Si
where Si and Sf correspond to nutrients concentration (in mg L
1) in
the beginning and at the end of cultivation time, respectively.
Nutrients uptake rates were determined by fitting the experi-
mental data, corresponding to the time-course evolution of nutri-
ents concentration, to the modified Gompertz model (Zwietering
et al., 1990). This model has already been used to describe micro-
algal (Çelekli et al., 2008; Lacerda et al., 2011; Queiroz et al., 2011)
and bacterial (Zwietering et al., 1990) growth and was here
(1) simplified to determine nutrients removal kinetics, as represented
in Equation (3):


SðtÞ ¼ Si þ Sf
Si $expð
exp½k$ðl
tÞ þ 1Þ (3)
where S(t) is the time-course evolution of nutrients (N, P or COD)
concentration, k is the nutrients uptake rate (in d
1) and l is the lag
time (in d). The kinetic parameters, k and l, were determined by
minimizing the sum of squared residuals using the Solver supple-
ment of Microsoft Excel 2013.
2.5. Statistical analysis
For each parameter, the average and the standard deviation
were calculated. The statistical significance of the results was
evaluated using the Student's paired t-test to investigate whether
the differences between the studied cultures could be considered
significant. This analysis was performed using the statistical soft-
ware SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Statistical tests were
carried out at a significance level of 0.05.
3. Results and discussion
3.1. Microalgal and bacterial growth in single cultures and in the
consortia
Fig. 1 shows the growth curves obtained for C. vulgaris and for
the three isolates when grown in single cultures (Fig. 1A, C and E)
 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357 351

Fig. 1. Growth curves obtained for the studied microorganisms when grown in single cultures (A, C and E) and in the developed consortia (B, D and F). Error bars correspond to the
standard deviation of the mean determined for four independent experiments.

and in the three studied consortia (Fig. 1B, D and F). Kinetic growth
parameters, such as maximum cell concentrations and average
biomass productivities, are shown in Table 1. Regarding the bac-
terial isolates, data from Fig. 1 evidence the typical growth phases
under batch conditions (Barsanti and Gualtieri, 2006; Monod,
1949): (i) adaptation or lag phase; (ii) acceleration phase; (iii)
exponential or log phase; (iv) retardation phase; (v) stationary
phase; and (vi) declining phase. Cultures of E. asburiae (Fig. 1A and
B) have experienced an exponential growth phase of approximately
24 h. At this stage the bacterial concentration started decreasing
until the end of cultivation time. Comparing the behaviour of this
isolate in single cultures and in the CE consortium, maximum cell
concentration achieved in single cultures ((11.9 ± 1.4)  108 CFU per
mL) was statistically higher (p ¼ 0.006) than maximum cell con-
centration achieved in the consortium ((8.70 ± 0.17)  108 CFU per
mL). Regarding Klebsiella sp. growth curves (Fig. 1C and D),

Table 1
Maximum cell concentrations (Nmax, 106 cells per mL or 108 CFU per mL) and average biomass productivities (P, g DW L
1 d
1) determined for the studied microorganisms
when grown in single cultures and in the developed consortia. (Values are presented as the mean ± standard deviation of four independent experiments; different letters (a, b
and c) within the same column represent statistically different values (p < 0.05); n.a. e not applicable; n.d. e not determined).

Microorganisms NM,max (106 cells per mL) NB,max (  108 CFU per mL) P (g DW L
1 d
1)
a
C. vulgaris 8.27 ± 0.79 n.a. 0.0214 ± 0.0040a
E. asburiae n.a. 11.9 ± 1.4a n.d.
Klebsiella sp. n.a. 6.15 ± 0.49b n.d.
R. ornithinolytica n.a. 15.1 ± 0.4c n.d.
CE 19.8 ± 2.0b 8.70 ± 0.17b 0.0359 ± 0.0018b
CK 8.74 ± 0.94a 7.20 ± 1.13b 0.0250 ± 0.0012a
CR 14.4 ± 2.3c 12.4 ± 0.1a 0.0314 ± 0.0030b
352 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357

exponential growth phase also lasted for approximately 24 h in
both cultures. After this time cell concentration of single cultures
was kept approximately constant (stationary growth phase) until
the third day of culturing, where cell concentration started
decreasing. In the CK consortium, an absence of the stationary
growth phase was observed and cell concentration started
decreasing after the first day of culturing. R. ornithinolytica growth
curves (Fig. 1E and F) have shown an exponential growth phase of
approximately one day, followed by a retardation phase until the
second day of culturing and then a declining phase until the end of
cultivation time. As it was observed for E. asburiae, maximum cell
concentration achieved by R. ornithinolytica in single cultures
((15.1 ± 0.4)  108 CFU per mL) was statistically higher (p ¼ 0.042)
than the one achieved in the CR consortium ((12.4 ± 0.1)  108 CFU
per mL). The short duration of exponential growth phase observed
for the bacteria is related to their higher specific growth rates, when
compared to those of microalgae. Additionally, since these studies
were performed in batch, the end of this growth phase is associated
to the decrease observed in soluble COD (Fig. 2E and F). Lower cell
concentrations achieved in the CE and CR consortia, as well as the
lack of a stationary growth phase until the third day of culturing for
Klebsiella sp. grown in the consortium, may be related to the
competition for the organic carbon source with C. vulgaris.
Although microalgal growth is mainly autotrophic, when both
organic and inorganic carbon sources are supplied, microalgae
perform both photosynthesis and oxidative assimilation (Cuellar-
Bermudez et al., 2015; Ogawa and Aiba, 1981). This assumption
can be confirmed through the decrease observed in soluble COD in
the single C. vulgaris culture (Fig. 2E). Regarding microalgal growth,
CE and CR consortia have shown a great influence on C. vulgaris
growth kinetics. Fig. 1 also shows the growth curves obtained for
C. vulgaris grown in single cultures and in the consortia. Analysis of
these growth curves shows that, in all conditions, C. vulgaris
experienced an adaptation phase that lasted approximately one
day, followed by the exponential growth phase. Duration of this
growth phase was strongly influenced by the presence of the co-
cultivated microorganisms: (i) in the single C. vulgaris culture
(Fig. 1A, C and E), this growth phase lasted for two days; (ii) in the
CE consortium (Fig. 1B) it lasted until the fourth day of culturing,
followed by a linear growth until the end of cultivation time; (iii) in
the CK consortium (Fig. 1D) it lasted for two days; and (iv) in the CR
consortium (Fig. 1F) it lasted for three days. Concerning maximum
cell concentrations (Table 1), co-cultivation of C. vulgaris with
E. asburiae and R. ornithinolytica has resulted in an increased
microalgal cell concentration. Maximum cell concentrations ach-
ieved in single cultures ((8.27 ± 0.79)  106 cells per mL) were
statistically lower (p < 0.05) than those determined for the CE
((19.8 ± 2.0)  106 cells per mL) and CR ((14.4 ± 2.3)  106 cells per
mL) consortia. Higher cell concentrations achieved in the CE con-
sortium were not surprising since bacteria from the genus

Fig. 2. Time-course evolution of nitrogen (A and B), phosphorus (C and D) and chemical oxygen demand (E and F) in the synthetic effluent determined for the studied micro-
organisms when grown in single cultures (A, C and E) and in the developed consortia (B, D and F). Error bars correspond to the standard deviation of the mean determined for four
independent experiments. The lines represent the model fit of the modified Gompertz model to the experimental data. The horizontal dotted lines correspond to the limits for
nutrients concentration in discharged effluents imposed by EU legislation (10 and 15 mg N L
1 for N, 1 and 2 mg P L
1 for P and 125 mg O2 L
1 for COD).
 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357
Enterobacter are known for their role as plant growth-promoting
bacteria (Bashan et al., 1993). Maximum cell concentration ach-
ieved in the CK consortium ((8.74 ± 0.94)  106 cells per mL) was
not statistically higher (p ¼ 0.176) than the one determined for
single C. vulgaris cultures. Statistically higher (p < 0.05) average
biomass productivities were also determined for CE and CR con-
sortia: 0.0359 ± 0.0018 and 0.0314 ± 0.0030 g DW L
1 d
1,
respectively. On the other hand, average biomass productivities
determined for the single C. vulgaris culture and for the CK con-
sortium were not statistically different (p ¼ 0.569).
These results suggest that co-cultivation of C. vulgaris with
E. asburiae and R. ornithinolytica improved microalgal growth. The
improvement in C. vulgaris growth is probably related to metabolic
cooperation established between the microalga and the co-
cultured bacterium. Previous studies have concluded that the
growth of C. vulgaris can be promoted by the presence of plant
growth-promoting bacteria, such as Azospirillum brasilense, Rhizo-
bium sp. and Bacillus licheniformis (De-Bashan et al., 2004; Kim
et al., 2014; Liang et al., 2013). Additionally, in the study per-
formed by Park et al. (2008), co-inoculation of Chlorella ellipsoidea
with eight bacterial strains isolated from a long-term culture of this
microalga resulted in microalgal growth increase of up to three
times compared to C. ellipsoidea alone.
3.2. Nutrients removal
Fig. 2 shows the time-course evolution of nitrogen (Fig. 2A and
B), phosphorus (Fig. 2C and D) and COD (Fig. 2E and F) present in
the synthetic effluent for single cultures of C. vulgaris and the
bacterial isolates (Fig. 2A, C and E) and for the consortia (Fig. 2B, D
and F).
From Fig. 2A it is possible to see that single C. vulgaris cultures
were able to effectively remove nitrogen from the synthetic
effluent, reaching concentrations below the limits imposed by EU
legislation at the end of cultivation time (0.495 ± 0.231 mg N L
1).
Regarding the bacterial isolates, Fig. 2A shows the ability of Kleb-
siella sp. and R. ornithinolytica to assimilate nitrate-nitrogen, being
R. ornithinolytica faster than Klebsiella sp. On the other hand,
E. asburiae was not able to remove nitrogen from the synthetic
effluent. At the end of cultivation time, nitrogen concentration
present in the synthetic effluent resulting from the single Klebsiella
sp. culture was 17.1 ± 0.3 mg N L
1, whereas nitrogen concentration
in the synthetic effluent resulting from the single R. ornithinolytica
culture was almost negligible. Nitrate-nitrogen removal by bacteria
was thought to be an anaerobic process conducted by denitrifying
bacteria. However, the presence of oxygen in the synthetic effluent
within the cultivation time (Fig. 3A and B) suggests another nitro-
gen removal mechanism. In fact, several studies have reported the
ability of some bacteria, especially from the genus Klebsiella, to
assimilate nitrate-nitrogen (Zhou et al., 2007). In this process, ni-
trate is consecutively reduced into nitrite and into ammonia, which
is then incorporated into carbon skeletons (Pin ~ ar et al., 1997, 1998;
Zhou et al., 2007). The studied consortia have also shown a great
ability for nitrogen removal, reaching concentrations very close to
0 mg N L
1 at the end of the experiments. Since the synthetic
medium used in this study did not present ammonia in its
composition, it is thought that the mechanism involved in nitrogen
removal by C. vulgaris was assimilation followed by reduction into
ammonia. Although both single and dual-species consortia of
C. vulgaris have achieved the limits for nitrogen concentration in
discharged effluents, it is possible to see that nitrogen uptake in the
studied consortia was faster than in the single C. vulgaris culture.
Load reduction percentages or removal efficiencies were also
determined for the studied cultures (Table 2). Nitrogen removal
efficiencies determined in this study ranged from 0 to 100%. Taking
353
into account the minimum percentage reduction established by EU
legislation, 70%, this value was achieved by the single
R. ornithinolytica and C. vulgaris cultures and by all studied con-
sortia. In a previous study conducted by Wang et al. (2010), a
nitrate-nitrogen removal of 62.5% was obtained when growing
Chlorella sp. in a wastewater collected from the secondary settling
tank of a MWTP located in Minnesota (USA). Looking at nutrients
removal profiles (Fig. 2), nutrients uptake by C. vulgaris and the
bacterial isolates grown in single cultures and in the developed
consortia goes through an adaptation phase, also known as lag
phase, followed by an exponential decrease and a stationary phase.
These are the typical phases of microbial nutrient consumption in
batch systems. Therefore, the modified Gompertz model (Equation
(3)), commonly applied to simulate microbial growth, was fitted to
the experimental data to determine the kinetic parameters asso-
ciated to nutrients uptake. The curved lines present in Fig. 2
represent the model fits of the modified Gompertz model to the
experimental data. Analysis of the coefficient of determination (R-
squared values close to one) indicates that the Gompertz model
correctly describes nutrients (nitrogen, phosphorus and COD) up-
take by the studied microorganisms and consortia. Looking at lag
time values determined for nitrogen uptake, it is possible to
conclude that single C. vulgaris, Klebsiella sp. and R. ornithinolytica
cultures, as well as the studied consortia presented a short lag time,
starting nitrogen uptake before completing the first day of
culturing. Regarding nitrogen uptake rates, the values obtained
through the modified Gompertz model ranged from 0.831 ± 0.122
to 5.61 ± 0.43 d
1. The lowest value was determined for the single
Klebsiella sp. culture. However, this value was not statistically
different from those determined for the single C. vulgaris culture
(1.42 ± 0.23 d
1; p ¼ 0.117) and for the CR consortium
(1.58 ± 0.16 d
1; p ¼ 0.083). Statistically higher values were
determined for the single R. ornithinolytica culture (5.48 ± 0.16 d
1;
p ¼ 0.001) and for the CE and CK consortia: 5.61 ± 0.43 d
1 (p ¼
0.019) and 3.32 ± 0.02 d
1 (p ¼ 0.002), respectively. Lower nitrate-
nitrogen removal rates (1.5 ± 0.3 d
1) were reported for C. vulgaris
grown in commercial synthetic Combo medium (Ruiz et al., 2013).
These results have shown that the CE and CK consortia have fav-
oured nitrogen uptake, resulting in increased nitrogen uptake rates
and in a reduction in the time required for the achievement of the
EU legislation limits for discharged effluents (Table 2). When grown
in single cultures, the first limit imposed by EU legislation was
reduced to, at least, half of the value determined for the single
C. vulgaris culture. When grown in single cultures, the first limit
imposed by legislation (15 mg N L
1) was reached after 33.1 h of
culturing, whereas the same value was reached after 11.9 and 17.2 h
in the CE and CK consortia, respectively. Although nitrate-nitrogen
concentrations after the second day of culturing were almost
negligible, microalgal growth in the CE and CR consortia proceeds
after the third day. These results may be due to the uncoupled
microalgal growth. According to the Droop (1968) model, which
assumes that microalgal growth depends on the intracellular car-
bon, nitrogen and phosphorus quotas instead of the extracellular
available nutrients, microalgal growth after nutrients depletion
(uncoupled growth) is possible to occur.
Regarding phosphorus uptake (Fig. 2C and D), C. vulgaris single
cultures and consortia were able to effectively remove this nutrient
from the synthetic effluent, reaching the limits defined by EU
legislation (2 mg P L
1 for a PE of 10e100 thousand and 1 mg P L
1
for a PE higher than 100 thousand). Since the pH of the synthetic
effluent in these cultures (Fig. 3C and D) has not exceeded 8, except
in the last days of culturing where no measurable quantity of
phosphorus was detected, phosphate precipitation is not expected
to occur. Accordingly, it can be proposed that the mechanism
involved in phosphorus removal is bioassimilation. Among the
354 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357

Fig. 3. Time-course evolution of dissolved oxygen concentration (A and B) and pH (C and D) in the synthetic effluent determined for the studied microorganisms when grown in
single cultures (A and C) and in the developed consortia (B and D). Error bars correspond to the standard deviation of the mean determined for four independent experiments.

Table 2
Nutrients (N, P and COD) removal efficiencies (%R), kinetic parameters of nutrients assimilation obtained through the modified Gompertz model (l, d and k, d
1) and time (t, h)
necessary to reach the limits established by EU legislation. (Values are presented as the mean ± standard deviation of four independent experiments; different letters (a, b and
c) within the same column, for the same nutrient, represent statistically different values (p < 0.05); n.a. e not applicable).

Microorganisms %R l (d) k (d
1) R2 t1 (h) t2 (h)

N C. vulgaris 99.8 ± 0.3a 0.135 ± 0.032a 1.42 ± 0.23a 0.991 33.1 41.5
E. asburiae n.a. n.a. n.a. n.a. n.a. n.a.
Klebsiella sp. 49.9 ± 1.2b 0.0851 ± 0.0203a 0.831 ± 0.122a 0.984 n.a. n.a.
R. ornithinolytica 99.5 ± 0.9a 0.688 ± 0.013b 5.48 ± 0.16b 1.000 9.62 11.7
CE 100 ± 0a 0.250 ± 0.084a 5.61 ± 0.43b 1.000 11.9 14.2
CK 97.0 ± 4.8a 0.146 ± 0.009a 3.32 ± 0.02c 0.992 17.2 21.1
CR 100 ± 0a 0.0549 ± 0.0359a 1.58 ± 0.16a 0.989 26.5 34.1

P C. vulgaris 95.9 ± 0.6a 0.650 ± 0.030a 1.56 ± 0.21a 0.997 56.8 72.8
E. asburiae n.a. n.a. n.a. n.a. n.a. n.a.
Klebsiella sp. n.a. n.a. n.a. n.a. n.a. n.a.
R. ornithinolytica 43.1 ± 4.7b 0.170 ± 0.021b 1.07 ± 0.01a 0.992 n.a. n.a.
CE 96.2 ± 1.4a 0.179 ± 0.033b 3.01 ± 0.28b 0.999 25.5 32.9
CK 95.6 ± 2.2a 0.281 ± 0.031b 4.02 ± 1.27b 1.000 22.9 26.6
CR 95.8 ± 2.1a 0.179 ± 0.010b 3.60 ± 0.15b 0.998 23.9 27.8

COD C. vulgaris 91.0 ± 8.3a 0.637 ± 0.152a 1.16 ± 0.64a 0.999 46.8 n.a.
E. asburiae 85.3 ± 5.1a 0.0427 ± 0.0183b 1.49 ± 0.53a 0.988 30.0 n.a.
Klebsiella sp. 95.2 ± 0.9a 0b 0.954 ± 0.066a 0.983 30.5 n.a.
R. ornithinolytica 87.9 ± 4.4a 0b 1.23 ± 0.11a 0.974 29.9 n.a.
CE 95.0 ± 0.7a 0.116 ± 0.054b 2.53 ± 0.52b 0.993 17.4 n.a.
CK 85.6 ± 3.8a 0.431 ± 0.204a 2.12 ± 0.40b 0.991 27.8 n.a.
CR 89.4 ± 2.9a 0.153 ± 0.007b 3.02 ± 0.02b 0.982 17.4 n.a.

bacterial isolates, only R. ornithinolytica was able to uptake phos-
phorus, however, with modest efficiencies (43.1 ± 4.7%). In this
culture, phosphorus concentration decreased from 9.25 ± 0.06 to
5.26 ± 0.22 mg P L
1 (in the third day of culturing), being constant
until the end of the experiments. The ceasing of phosphorus uptake
at third day is apparently related to the declining growth phase
experienced by R. ornithinolytica after the second day of culturing.
The assimilation and storage of phosphate in the form of poly-
phosphates has already been reported for several microorganisms,
known as polyphosphate accumulating microorganisms (Mino
et al., 1998; Zafiriadis et al., 2012). However, the low removal effi-
ciencies demonstrated by the bacterial isolates may be related to
the low ability of activated sludge for phosphorus uptake (Su et al.,
2012). Comparing C. vulgaris cultures, it is clear that phosphorus
uptake by the three studied consortia was faster than phosphorus
uptake determined for single C. vulgaris cultures. Regarding phos-
phorus removal efficiencies, values determined for all C. vulgaris
cultures were not statistically different (p > 0.05), ranging from
95.6 ± 2.2 to 96.2 ± 1.4%. In these conditions, the minimum
reduction percentage of 80% established by EU legislation was
 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357
achieved. Similar removal percentages were obtained by Wang
et al. (2010) when culturing Chlorella sp. in a wastewater
collected before and after primary settling: 83.2 and 90.6%,
respectively. As well as for nitrogen, phosphorus uptake rates were
determined by fitting the modified Gompertz model to the exper-
imental data. Table 2 presents values of lag time, uptake rates, co-
efficient of determination given by this model and time required to
achieve the limits for phosphorus concentration in discharged ef-
fluents established by EU legislation. Similar lag time values
(p > 0.05) were determined for both nitrogen and phosphorus
uptake, indicating that both nutrients were readily assimilated (lag
time values lower than 1 d). Comparing single C. vulgaris cultures
with the studied consortia, a statistically higher lag time (p < 0.05)
was determined for single C. vulgaris cultures (0.650 ± 0.030 d). In
the CE, CK and CR consortia, lag time values were 0.179 ± 0.033,
0.281 ± 0.031 and 0.179 ± 0.010 d, respectively. In the case of
phosphorus uptake rates, a statistically lower value (p < 0.05) was
determined for the single C. vulgaris culture: 1.56 ± 0.21 d
1. When
cultured with E. asburiae, Klebsiella sp. and R. ornithinolytica,
phosphorus uptake rates increased to 3.01 ± 0.28, 4.02 ± 1.27 and
3.60 ± 0.15 d
1, respectively. Phosphorus uptake rates in the same
order of magnitude were obtained for C. vulgaris in the study per-
formed by Ruiz et al. (2013): when growing C. vulgaris in different
synthetic media and different wastewaters, phosphorus removal
rates ranged from 2.0 ± 0.2 to 8.7 ± 1.2 d
1. The reduction of lag
times, as well as the increase in phosphorus uptake rates deter-
mined for dual-species consortia, have contributed to a decrease in
the time required for the achievement of EU legislation limits. In
single C. vulgaris cultures the time required for the achievement of
the first limit established for phosphorus was 56.8 h, whereas in the
CE, CK and CR consortia this value decreased to 25.5, 22.9 and
23.9 h, respectively. The almost complete depletion of phosphorus
after the second day of culturing and the increase in microalgal cell
concentration in the CE and CR consortia reinforces the idea that
uncoupled microalgal growth may have occurred, as proposed by
Droop (1968). The increase in phosphorus uptake rates observed
between single C. vulgaris cultures and the CK consortium, as well
as the absence of effect of Klebsiella sp. on microalgal growth sug-
gests that in this consortium luxury uptake of phosphorus might
have occurred. According to Powell et al. (2008, 2009), microalgae
can adopt this assimilation mechanism, which consists in the up-
take of nutrients for storage within the biomass rather than
biomass production.
In terms of organic matter present in the synthetic effluent, all
studied cultures have effectively removed soluble COD (Fig. 2E and
F), reaching the limit defined by EU legislation (125 mg O2 L
1).
From Fig. 2E it is possible to see that soluble COD present in the
synthetic effluent corresponding to the bacterial isolates reached
very low concentrations at the third day of culturing, which is in
accordance with the decrease in biomass concentrations observed
in single cultures of these bacteria (Fig. 1A, C and E). COD removal in
the single C. vulgaris culture (Fig. 2E) indicates that microalgal
growth in these conditions was mixotrophic. Additionally, it is
possible to see from Fig. 2E and F that the supplied organic carbon
source was almost eliminated within the first three days in the
studied consortia and within the first five days in the single
C. vulgaris culture. A complete COD removal was not observed in
this study may be because of the detection limit of the analytical
method used for COD determinations. These results indicate that
after the third and fifth days, the concentration of organic carbon
was very low. However, the low concentrations of organic carbon
have not limited microalgal growth, especially in the CE and CR
consortia, where exponential growth phase lasted for longer pe-
riods of time (Fig. 1B and F). One possible reason for these results is
the ability of microalgae to perform photosynthesis, using CO2
355
(supplied in the air stream bubbled into the cultures) as carbon
source (Su et al., 2011). At the end of the experiments, similar COD
removal percentages (Table 2) were obtained in all studied cultures,
ranging from 85.3 ± 5.1 to 95.0 ± 0.7%. Similar removal percentages
(approximately 90%) were obtained in the study performed by Li
et al. (2011) when culturing Chlorella sp. in a domestic waste-
water. Similarly, a COD removal percentage of 98% was obtained by
Su et al. (2011) when culturing a wastewater-born algal-bacterial
culture in a domestic wastewater. High removal efficiencies
determined for the studied cultures may be related to the high
ability of the selected microorganisms to use organic carbon, which
was reflected in the low lag time values obtained through the
modified Gompertz model (Table 2). However, it was observed that
the studied consortia have reached the limit imposed by EU legis-
lation before the single cultures of the studied microorganisms.
This observation was confirmed by the COD uptake rates obtained
through the modified Gompertz model (Table 2). From Table 2 it is
possible to see that COD uptake rates determined for the single
cultures were not statistically different (p > 0.05), ranging from
0.954 ± 0.066 to 1.49 ± 0.53 d
1. On the other hand, statistically
higher (p < 0.05) values were determined for the CE, CK and CR
consortia, ranging from 2.12 ± 0.40 to 3.02 ± 0.02 d
1. Therefore,
the time required for the achievement of EU legislation limits was
significantly reduced in the studied consortia. Comparing
C. vulgaris cultures, this value decreased from 46.8 h (in single
cultures) to 17.4, 27.8 and 17.4 h (in the CE, CK and CR consortia,
respectively). Higher uptake rates determined for the studied
consortia suggest a better performance of mixed cultures in COD
removal. These results are in accordance with the study performed
by Su et al. (2012), which showed that COD removal efficiencies of
single algal cultures (66.0 ± 6.0%) were lower than those deter-
mined for different algae:sludge ratios (91.2 ± 1.7e96.2 ± 1.1%). The
lower ability of single-cultured microalgae for COD removal may be
related to the absence of heterotrophic bacteria to enhance organic
carbon degradation (Su et al., 2012).
In general, nutrients uptake rates determined for the developed
consortia were higher than those determined for the single cultures
used as control, evidencing the synergistic effect of microalgal-
bacterial consortia. These results suggest that the studied consor-
tia improve nutrients removal kinetics, which is in agreement with
the determined kinetic growth parameters. Microalgae and cya-
nobacteria require high amounts of nitrogen and phosphorus for
proteins, which account for 40e60% of cell dry weight, nucleic acids
and phospholipids synthesis (Silva-Benavides and Torzillo, 2012),
meaning that an increase in microalgal growth may result in an
increased assimilation of both nitrogen and phosphorus. The
improvement of nutrients uptake kinetics in the studied consortia
may be related to the direct or indirect effect of the presence of
bacterial isolates in the studied consortia. On the one hand, nutri-
ents uptake rates may be higher due to the direct uptake promoted
by co-cultured isolates. This is the case of nitrogen uptake in the CK
and CR consortia, phosphorus uptake in the CR consortium and
COD uptake in the three studied consortia. On the other hand, the
presence of bacterial isolates in the studied consortia may have
resulted in the release of CO2 and other metabolites to the culture
medium, positively affecting microalgal growth. The excretion of
CO2 to the culture medium is considered one of the most important
factors contributing to the success of microalgal-bacterial cultures,
especially in wastewater treatment processes (Su et al., 2011, 2012).
Additionally, it has already been reported that bacteria supply
microalgae with organic growth factors and vitamins that enhance
microalgal growth (Bordel et al., 2009; Natrah et al., 2014; Unnithan
et al., 2014). The increase in nutrients uptake presents the advan-
tages of reducing the requirements for large bioreactors and also
hydraulic residence times. Reducing bioreactor dimensions and
356 A.L. Gonçalves et al. / Journal of Cleaner Production 133 (2016) 348e357
hydraulic residence times are important factors to consider in the
wastewater polishing, since operational costs can be significantly
reduced. The use of microalgal-based treating systems may also
contribute to the reduction of carbon emissions associated to
wastewater treatment plants, contributing to the development of a
near zero CO2 emissions process. However, for an application of
these microalgal-bacterial consortia in a MWTP, the presence of
contaminations should be avoided. For this, the following options
must be taken into account (Benemann et al., 1980; Mun ~ oz and
Guieysse, 2006; Wood, 1987): (i) selection of a fast growing and
highly resistant microalgae, such as Chlorella or Scenedesmus; and
(ii) manipulation of the operational conditions, such as hydraulic
residence times and recirculation of biomass to sustain specific
microalgal populations.
4. Conclusions
This work has shown that consortia of C. vulgaris with selected
bacterial isolates from a MWTP resulted in a synergistic relation-
ship between these microorganisms, increasing biomass produc-
tion and nutrients removal from a synthetic secondary-treated
effluent. After seven days of culturing, cell concentration of
C. vulgaris in the CE and CR consortia was, respectively, 58 and 42%
higher than the one determined for the single C. vulgaris culture.
Additionally, the three studied consortia have contributed to the
increase observed in nutrients (nitrogen, phosphorus and COD)
uptake rates, resulting in a significant reduction in the time
required for the achievement of EU legislation limits. In the studied
consortia, the time required by the single C. vulgaris culture to
achieve the established limits was reduced to, at least, half of its
value. Therefore, it can be concluded that the studied consortia (CE,
CK and CR) can be a promising alternative in wastewater polishing,
playing an important role in the reduction of bioreactor dimensions
and hydraulic residence times. These findings also contribute to the
production of microalgae within the biorefinery concept (an inte-
grated system of biomass conversion into transportation fuels,
power and chemicals).
Acknowledgements
This work was financially supported by: Project POCI-01-0145-
FEDER-006939 - Laboratory for Process Engineering, Environment,
Biotechnology and Energy e LEPABE e funded by FEDER funds
through COMPETE2020 e Programa Operacional Competitividade e
~o (POCI) e and by national funds through FCT e
Internacionalizaça
Fundaçaeo para a Ciencia e a Tecnologia e Project Novomar (ref.
0687 Novomar-1-P) and Scholarships SFRH/BD/88799/2012 and
SFRH/BPD/66721/2009.
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