J Appl Phycol (2012) 24:267–276
DOI 10.1007/s10811-011-9675-2
Nitrogen and phosphorus removal through laboratory batch
cultures of microalga Chlorella vulgaris and cyanobacterium
Planktothrix isothrix grown as monoalgal and as co-cultures
Ana Margarita Silva-Benavides & Giuseppe Torzillo
Received: 14 January 2011 / Revised and accepted: 24 March 2011 / Published online: 15 April 2011
# Springer Science+Business Media B.V. 2011
Abstract Batch cultures of the green microalga Chlorella
vulgaris and cyanobacterium Planktothrix isothrix and
their corresponding co-cultures were grown in municipal
wastewater in order to study their growth as well as the
nitrogen (NH4–N) and phosphorus (PO43−–P) removal.
The cultures were grown under two irradiances of 20 and
60 μmol photons m−2 s−1 in shaken and unshaken conditions.
The co-culture of unshaken Chlorella and Planktothrix
showed the greatest growth under both irradiances. The
monoalgal Planktotrix cultures showed better growth when
unshaken than when shaken, whereas Chlorella cultures grew
better when mixed, but only at the higher irradiance. The
highest percentage of nitrogen removal (up to 80%) was
attained by the unshaken co-cultures of Chlorella and
Planktothrix. The amount of nitrogen recycled in the biomass
reached up to 85% of that removed. Shaken monoalgal
cultures of Chlorella showed phosphorus removal under both
irradiances. They completely removed the initial phospho-
rus concentration (7.47±0.17 mg L−1) within 96 and 48 h
under 20 and 60 μmol photons m−2 s−1, respectively.
A. M. Silva-Benavides
Escuela de Biología, Universidad de Costa Rica,
San Pedro, San José 2060, Costa Rica
A. M. Silva-Benavides
Centro de Investigación en Ciencias del Mar y Limnología
(CIMAR), Ciudad de la Investigación, Universidad de Costa Rica,
San Pedro, San José 2060, Costa Rica
G. Torzillo (*)
Istituto per lo Studio degli Ecosistemi del CNR, Sede di Firenze,
via Madonna del Piano, 10,
50019, Sesto Fiorentino, Firenze, Italy
e-mail: torzillo@ise.cnr.it
Keywords Chlorella . Planktothrix . Co-cultures . Nitrogen
and phosphorus removal . Wastewater treatment
Introduction
One of the advantages of using microalgae in tertiary
wastewater treatment systems is the possibility of recycling
the biomass into potentially valuable products, such as
fertilisers and fine chemicals, thus helping to meet the
overall costs of the treatment plant (de la Noüe and Pauw
1988; de la Noüe et al. 1992; de-Bashan and Bashan 2004).
Microalgae grown on wastewater for energy production
have also been proposed for a long time (Benemann et al.
1977; Oswald and Golueke 1960). However, in recent
years, because of global warming and depletion of fossil
fuel and the need for mitigation of greenhouse gas
emission, the exploitation of microalgae in phycoremedia-
tion of municipal wastewater coupled to biomass produc-
tion for biofuel extraction is more feasible (Rawat et al.
2011; Park et al. 2011).
Microalgae can be efficiently used to remove significant
amounts of nutrients because they require high amounts of
N and P for proteins (40–60% of dry weight), nucleic acid
and phospholipids synthesis. Nutrient removal from waste-
water treated with microalgae is further enhanced through
NH3 stripping and P deposition due to the raise of pH
associated with photosynthesis (Oswald 2003; Laliberté et
al. 1994; Larsdotter 2006a). However, atmospheric deposi-
tion of reactive nitrogen has been well documented to be a
source of nutrient enrichment and one of the sources of
acidification that can have deleterious impacts on terrestrial
and aquatic ecosystems (Driscoll et al. 2001; Pinder et al.
2008). The increasing environmental concern is a stimulus
268
towards the application of more sustainable wastewater
treatment systems. In order to reduce the emission of NH3
to atmosphere, the control of the pH of the culture is
therefore mandatory (Park and Craggs 2010).
Several studies have been dedicated to the use of single
species of microalgae (Tam and Wong 1996; Voltolina et al.
2005; de-Bashan et al. 2007) and cyanobacteria (Pouliot et
al. 1989; de la Nüe et al. 1993; Chevalier et al. 2000;
Olguín et al. 2003) for wastewater applications. However,
the notion that a combination of more than one
microorganism is better than a single organism is gaining
acceptance. For example, when grown as a monoculture,
neither Rhodopseudomonas sphaeroides nor Chlorella
sorokiniana could simultaneously remove propionate,
ammonia, nitrate and phosphate from synthetic wastewater,
whilst a mixed culture could accomplish it (Ogbonna et al.
2000). Chlorella and a macrophyte (Lemna minuscola) could
be applied in tandem for the biological treatment of
recalcitrant aerobic industrial effluent, wastewater that
otherwise would have prevented the growth of any macro-
phyte (Valderrana et al. 2002). Relatively little information is
available on the use of cyanobacteria and microalgae grown
in mixed cultures (co-culture). Their substantial diversity in
pigment antenna composition (i.e. absorption spectra) and
the presence (e.g. Planktothrix) and absence (Chlorella) of
gas vacuoles can be useful for improving the light
conversion to biomass and the amount of nutrients removed
since these species can colonise different depths of the water
column in unstirred wastewater oxidation ponds.
The aim of this study was to investigate the growth of
cultures as well as the removal of nitrogen and phosphorus
from municipal wastewater that had previously been
subjected to a conventional secondary treatment by means
of the microalga Chlorella vulgaris and the cyanobacterium
Planktothrix isothrix and their corresponding co-cultures. In
order to limit ammonia loss to the atmosphere and
phosphorus deposition, the cultures were grown in atmo-
sphere enriched with CO2 and the pH was controlled daily.
The experimental design mainly aimed at achieving a high
recovery of nutrients. In order to examine the effect of
mixing on growth and on the efficiency of nitrogen and
phosphorus removal, shaken and unshaken cultures were
compared under two different light intensities.
Materials and methods
The planktonic green alga Chlorella vulgaris and the
cyanobacterium Planktothrix isothrix were isolated from
wastewater samples taken from secondary treatment oxida-
tion ponds operated by the Instituto Costarricense de
Acueductos y Alcantarillados of the town of Liberia, province
of Guanacaste, Costa Rica. Cells were grown in a BG11
J Appl Phycol (2012) 24:267–276
medium (Rippka et al. 1979) in glass columns with a 400-mL
working volume, at 28°C, and bubbled with a mixture of air/
CO2 (97:3, v/v). Cultures were illuminated with cool white
fluorescent lights (Dulux L, 55W/840, Osram, Italy), which
provided 20 μmol photons m−2 s−1 on the surfaces of the
culture.
The culture medium used was a treated effluent from a
conventional secondary treatment facility supplied by the
Municipality of Florence (Italy). The effluent was first
centrifuged to remove solid particles and was then frozen to
preserve the original composition. After thawing, the samples
were sterilised by means of filtration using sterile 0.45-μm
pore size membranes (Sartorius, Germany). The experiments
were carried out with monoalgal batch cultures of C. vulgaris
and P. isothrix and also with a mixture of both species. For
the experiments, log phase cells were harvested by centrifu-
gation and then resuspended in filtered sterile distilled water
(three times) to eliminate any traces of BG11 nutrients.
Experimental design
The experiments were carried out in triplicate 250 mL
cultures in 500-mL Erlenmeyer flasks. All experiments
started with a mean initial dry weight of 190 mg L−1.
Cultures were incubated at a constant temperature of 28°C
in an orbital incubator (New Brunswick Scientific, USA)
under 20 and 60 μmol photons m−2 s−1 measured on the
surface of the cultures with a corrected cosine flat sensor
(Licor quantum). All experiments were carried out under
continuous illumination. In order to evaluate the effect of
mixing, shaken (80 oscillations min−1) and unshaken
cultures were compared. To provide sufficient CO2 for
growth, the atmosphere of the incubator chamber was
flushed with a mixture of air/CO2 (97:3, v/v) at a
continuous flow rate of 5 Lmin−1. NH4Cl and K2HPO4
were added to the effluent in order to attain the same N and
P concentrations as those of the oxidation ponds of Liberia
(Costa Rica), i.e. NH4–N=79.3 mg L−1 and PO43−–P=
7.47 mg L−1. The initial pH of the cultures was 7.0, and this
was adjusted daily to the initial value by adding a diluted
sterile HCl.
Analytical methods
Samples were taken at time intervals to determine the P and
N concentrations and the dry mass. Cell dry weight
determination was performed on triplicate 10-mL samples.
Each sample was filtered through 0.7-μm pore size pre-
weighted filters (Whatman grade GF/F) and then dried at
105°C for 3 h.
PO43−–P and NH4–N concentrations were measured on a
Hanna Instruments (USA) Multiparameter Bench Photometer
J Appl Phycol (2012) 24:267–276 269

(model C99), in accordance with the Standard Methods for the
Examination of Water and Wastewater (APHA 1992). When
measurements were carried out, care was taken to avoid any
loss of gaseous ammonia. Samples were acidified and then
centrifuged at 7,860×g for 5 min in order to separate the
algal cells. To track ammonia loss due to degassing, controls
(three replicates) without cells were maintained under the
same conditions as in the experiments with cells. The N
content of the biomass was measured using a CHNS-O
Analyser (model Flash EA 1112 series, Thermo Electron
Corporation, the Netherlands).
Biomass productivity and NH4–N and PO43−–P removal
were calculated from the difference between the final and
initial values. To facilitate a comparison of the capacity for
P removals among the cultures, the halftime (t1/2) for
attaining a 50% reduction in the initial phosphorus
concentration was calculated by drawing a vertical line to
the abscissa axis originating from the intersection of a
horizontal line (corresponding to 50% PO43−–P initial
concentration) and the fitting curves of phosphorus con-
centrations. All experiments were repeated three times on
independently grown cultures.
Statistical analysis
All statistical tests were carried out using a statistical
software package (Stat graphics Plus, version 5.1 for
Windows). The differences in all the data were tested by
means of a two-way ANOVA using Duncan test of variance
at a 95% confidence interval.
Results
Culture productivities at 20 μmol photons m−2 s−1
During the course of the experiments, growth of the
cultures increased linearly. Under this light irradiance,
significant differences (p=0.05) were found between strain
productivities and between shaken and unshaken cultures
(Table 1). The highest interaction effect of strains and the
mixing regime on productivity was observed in unshaken
co-cultures of Chlorella and Planktothrix, very likely the
result of a higher growth contribution of Planktothrix.
Indeed, this cyanobacterium, which has gas vacuoles, could
float on the surface of the culture and thus benefit from a
better exposure to light.
Culture productivities at 60 μmol photons m−2 s−1
When the cultures were exposed to higher irradiance, the
productivities of both the shaken and the unshaken cultures
increased. The culture growth followed a linear pattern.
Significant differences (p =0.05) were found between
productivities of co-cultures (Chlorella and Planktothrix)
and monoalgal cultures of Chlorella and Planktothrix,

Table 1 Mean productivities

obtained with cultures of Growth conditions Biomass productivity (mg L−1 day−1)
Chlorella, Planktothrix and their
corresponding co-cultures 20 μmol photons m−2 s−1) 60 μmol photons m−2 s−1
grown under continuous light in
shaken and unshaken conditions Strains
Chlorella 30.54 c 42.09 b
Chlorella+Planktothrix 37.77 a 62.21 a
Planktothrix 34.90 b 41.64 b
Mixing regime
Shaken 28.12 b 48.78 n.s.
Unshaken 40.68 a 48.51 n.s.
Strains × mixing regime
Chlorella shaken 31.07 (1.11) 54.18 (0.23)
Chlorella unshaken 30.00 (2.22) 30.00 (0.50)
Chlorella+Planktothrix shaken 25.54 (1.13) 58.89 (1.11)
Chlorella+Planktothrix unshaken 50.00 (0.97) 65.54 (3.33)
Planktothrix shaken 27.75 (1.11) 33.29 (0.24)
Standard deviations are in Planktothrix unshaken 42.03 (0.85) 50.00 (1.11)
parenthesis (n=3). The values
for each parameter in the same Two-way ANOVA (p values)
column followed by different Strains 0.000 0.000
letters are significantly different Mixing regime 0.000 0.710
(p=0.05)
Strains × mixing regime 0.000 0.000
ns no significant differences
270 J Appl Phycol (2012) 24:267–276

whilst no significant differences were observed between
shaken and unshaken cultures (Table 1). The interaction
between the strains and the mixing regime resulted in a
higher effect in the unshaken co-cultures of Chlorella and
Planktothrix, which translated into a higher productivity
(65.5 mg L−1 day−1).
Nitrogen removal at 20 μmol photons m−2 s−1
The pH of the cultures, which was adjusted every day to
pH 7.0, ranged between 7.0 and 7.8 in the cultures and
between 7.0 and 7.4 in the control (without algae).
Significant differences (p=0.05) were found in N removal
between the strains and between the two mixing regimes
(shaken or unshaken; Table 2). At this irradiance, the
highest N removal rates were observed in the unshaken
cultures of Chlorella plus Planktothrix (about 62 mg L−1)
and in a monoculture of Planktothrix (59 mg L−1).
Figure 1a, b shows the kinetics of nitrogen removal for
both shaken and unshaken cultures grown under an
irradiance of 20 μmol photons m−2 s−1. As soon as the
cells were resuspended in an NH4–N-replete medium, an
appreciable decrease in its concentration was observed
(between 3% and 13.7% of the initial NH4–N). In the
shaken cultures, Chlorella and the co-culture of Chlorella
and Planktothrix reduced the initial NH4–N to approxi-
mately the same level (to 44% of the initial concentration).
Instead, within the same time interval (216 h), the
cyanobacterium Planktothrix reduced the amount to 56%
of the initial concentration (Fig. 1a). The mean loss of
ammoniacal N by degassing in the shaken control was
5.2% of the initial value.
With the unshaken cultures, the removal of N was
significantly higher as compared to that of the shaken ones
(Fig. 2b), except for Chlorella cultures which showed
approximately the same nitrogen removal capacity as the
shaken ones (Fig. 1b). Loss of ammoniacal N in the
unshaken control was found to be 6.7% of the initial
concentration.
Nitrogen removal at 60 μmol photons m−2 s−1
Under this irradiance, significant differences (p=0.05) were
recorded between strains, whilst differences between the
two mixing regimes resulted not significant (Table 2). The
highest N removals were achieved with unshaken co-
cultures of Chlorella and Planktothrix (64.6 mg L−1) and
with shaken Chlorella cultures (56.03 mg L−1).
The kinetics of N removals in shaken cultures resulted
very similar among the three cultures (Fig. 2a); nitrogen
concentration dropped to a mean of 34% of the initial value
after 216 h of culture loss of ammoniacal N by degassing,
as recorded in the control, which accounted for 6.6% of the
initial concentration. Unshaken cultures of Chlorella reduced
the initial concentration of NH4–N to 52%, whilst with
Planktothrix it was reduced to 32%: Planktothrix and
Chlorella co-cultures reduced it by 19% (Fig. 2b). Loss of
ammoniacal N accounted for 8.7% of the initial value.

Table 2 Total ammonia

nitrogen removal achieved Growth conditions Total ammonia nitrogen removal (mg L−1)
with cultures of Chlorella,
Planktothrix and their 20 μmol photons m−2 s−1) 60 μmol photons m−2 s−1
corresponding co-cultures
grown under continuous light in Strains
shaken and unshaken conditions Chlorella 39.65 c 47.05 c
Chlorella+Planktothrix 48.21 b 58.52 a
Planktothrix 51.54 a 51.42 b
Mixing regime
Shaken 37.42 b 52.21 n.s.
Unshaken 55.51 a 52.44 n.s.
Strains × mixing regime
Chlorella shaken 33.67 (0.35) 56.03 (0.81)
Chlorella unshaken 32.42 (0.51) 38.07 (0.08)
Chlorella+Planktothrix shaken 34.58 (0.63) 52.44 (0.78)
Standard deviations are in Chlorella+Planktothrix unshaken 61.85 (0.47) 64.59 (1.02)
parenthesis (n=3). The values Planktothrix shaken 44.02 (1.19) 48.17 (0.15)
for each parameter in the same Planktothrix unshaken 59.05 (0.10) 54.67 (0.94)
column followed by different
letters are significantly different Two-way ANOVA (p values)
(p=0.05). Mean initial ammonia Strains 0.000 0.000
nitrogen concentration was Mixing regime 0.000 0.520
79.3±1.47 mg L1
Strains × mixing regime 0.000 0.000
ns no significant differences
J Appl Phycol (2012) 24:267–276 271

in the initial amount of phosphorus in shaken monoalgal

cultures of Chlorella was 14 h, whilst it increased to 30–
42 h in the co-culture of Chlorella and Planktothrix and the
monoalgal Planktothrix cultures, respectively (Fig. 3a).
Among these cultures, Chlorella removed P almost com-
pletely (98% of the initial amount) within 96 h, whilst the
co-culture of Chlorella and Planktothrix took about 168 h.
In monoalgal Planktothrix cultures, the concentration of P
decreased to 33% of the initial value within 96 h and
remained constant thereafter (Fig. 3a).
In the unshaken cultures, the t1/2 increased to 96 h with
Chlorella, to 66 h with the co-culture of Chlorella and
Planktothrix, and to 144 h with cultures of Planktothrix
(Fig. 3b). Among unshaken cultures, only the co-culture of
Chlorella and Planktothrix removed phosphorus in its totality

Phosphorus removal at 60 μmol photons m−2 s−1

Fig. 1 NH4–N removals by C. vulgaris, P. isothrix and co-cultures of Under this irradiance, a significant difference (p=0.05) in
Planktothrix and Chlorella grown at 20 μmol photons m−2 s−1 in P removal was observed only between Planktothrix and
shaken (a) and unshaken conditions (b). Chlorella (circle), co-culture the other two cultures (Chlorella and co-cultures of
of Planktothrix and Chlorella (triangle), Planktothrix (inverted
triangle), control (without cells, diamond). Data were fitted with a
Planktothrix and Chlorella); significant differences were
one-phase exponential decay curve found between the two mixing regimes (Table 4). Under
higher irradiance, the kinetics of P removal was faster than
that observed with lower light (Fig. 4a, b). Indeed, in the
Nitrogen budget shaken cultures, a 50% reduction was achieved within 8 h
for the monoalgal Chlorella cultures, 16 h for the co-
The percentage of N in the biomass varied depending on
the organisms and the culture conditions, from 8.5% of dry
weight in Chlorella biomass to as much as 10.6% in
Planktothrix grown under low light conditions (Table 3).
Under 20 μmol photons m−2 s−1, the highest percentage of
NH4–N recycled in the biomass was attained with unshaken
cultures (average 70% of the removed amount). In the
cultures grown at 60 μmol photons m−2 s−1, the highest
percentage of NH4–N recycling was attained with either
shaken (85.8%) or not shaken (75.5%) co-cultures of
Chlorella and Planktothrix (Table 3).

Phosphorus removal at 20 μmol photons m−2 s−1

Under this irradiance, significant differences (p=0.05) in P

removal were found between strains and between shaken
and unshaken cultures. The highest P removal were attained
with shaken Chlorella cultures and with both shaken and
unshaken co-cultures, which totally removed the initial P,
whilst the lowest removal was attained with shaken cultures
of Planktothrix (Table 4).
Figure shows a comparison of the P removal kinetics in Fig. 2 NH4–N removal by C. vulgaris, P. isothrix and co-cultures of
cultures grown at 20 μmol photons m−2 s−1. As soon as the Planktothrix and Chlorella grown at 60 μmol photons m−2 s−1 in
shaken (a) and unshaken conditions (b). Chlorella (circle), co-culture
cells were resuspended in the replete phosphorus medium, a
of Planktothrix and Chlorella (triangle), Planktothrix (inverted
drop of between 2.6% and 7% of the initial value of P was triangle), control (without cells, diamond). Data were fitted with a
observed. The halftime (t1/2) for obtaining a 50% reduction one-phase exponential decay curve
272 J Appl Phycol (2012) 24:267–276

Table 3 Nitrogen budget of C. vulgaris, P. isothrix and their corresponding co-cultures grown under continuous light in shaken and unshaken
conditions

Organism and light irradiance NH4–N removal

Shaken Unshaken

Total NH4–N N (%) (nitrogen NH4–N recycled Total NH4–N N (%) (nitrogen NH4–N recycled
removal (mg L−1) content in the in the biomass removal (mg L−1) content in the in the biomass
biomass (mg L−1) biomass (mg L−1)

20 μmol photons m−2 s−1

C. vulgaris 33.6 8.5 23.8 32.4 8.5 23.0
C. vulgaris+P. isothrix 34.6 9.4 21.6 61.8 9.7 43.6
P. isothrix 43.6 10.6 26.5 59.0 10.6 40.3
60 μmol photons m−2 s−1
C. vulgaris 56.0 8.5 41.6 38.0 8.5 23.0
C. vulgaris+P. isothrix 52.2 8.4 44.8 65.0 8.4 49.7
P. isothrix 48.1 8.4 25.2 54.0 8.4 37.8

culture of Planktothrix and Chlorella, and 44 h for the Planktothrix removed as much as 83% of the initial
monoalgal cultures of Planktothrix (Fig. 4a). Complete concentration (Fig. 4b).
phosphorus removal occurred after 48 h with Chlorella,
after 96 h with the co-culture and after 216 h with
Planktothrix (Fig. 4a). The corresponding t1/2 for the Discussion
unshaken cultures was 21 h for Chlorella, 34 h for co-
cultures of Chlorella and Planktothrix, and 52 h for Culture growth
Planktothrix (Fig. 4b). Complete removal of P was
achieved with Chlorella after 120 h and by the co- To achieve the desired level of water treatment with algal
culture after 168 h, whilst under these conditions, systems, optimising autotrophic growth is mandatory, and

Table 4 Total phosphorus

removal achieved with cultures Growth conditions Total phosphorus removal (mg L−1)
of Chlorella, Planktothrix and
their corresponding co-cultures 20 μmol photons m−2 s−1 60 μmol photons m−2 s−1
growth under continuous light in
shaken and unshaken conditions Strains
Chlorella 6.95 b 7.43 a
Chlorella+Planktothrix 7.52 a 7.45 a
Planktothrix 5.16 c 6.82 b
Mixing regime
Shaken 6.48 b 7.52 a
Unshaken 6.61 a 6.95 b
Strains × mixing regime
Chorella shaken 7.35 (0.08) 7.52 (0.14)
Chlorella unshaken 6.54 (0.05) 7.35 (0.05)
Chlorella+Planktothrix shaken 7.35 (0.08) 7.55 (0.05)
Chlorella+Planktothrix unshaken 7.40 (0.10) 7.35 (0.03)
Standard deviations are in Planktothrix shaken 4.73 (0.06) 7.50 (0.10)
parenthesis (n=3). The values Planktothrix unshaken 5.59 (0.03) 6.15 (0.05)
for each parameter in the same Two-way ANOVA (p values)
column followed by different
letters are significantly different Strains 0.000 0.000
(p=0.05). Mean initial phos- Mixing regime 0.002 0.000
phorus concentration was Strains × mixing regime 0.000 0.000
7.47±0.17 mg L−1
J Appl Phycol (2012) 24:267–276
Fig. 3 PO43−–P removal by C. vulgaris, P. isothrix and co-culture of
Planktothrix and Chlorella grown at 20 μmol photons m−2 s−1 in
shaken (a) and unshaken conditions (b). Chlorella (circle), co-culture
of Planktothrix and Chlorella (triangle), Planktothrix (inverted
triangle), control (without cells, diamond). Data were fitted with a
one-phase exponential decay curve. Broken line indicates a 50%
reduction in the initial phosphorus concentration
basic principles of algal mass cultures must be applied
(Goldman 1979; Torzillo et al. 2003). Light availability and
light/dark regime represent two important factors affecting
productivity. The latter is strongly influenced by the
mixing. In addition, mixing prevents: (1) sedimentation
and thermal stratification, (2) the formation of nutrients and
pH gradients, (3) depletion of carbon dioxide on the pond
surface and (4) the improvement of the stripping of
supersaturating oxygen (Richmond 2003). However, the
effect of mixing on a light/dark regime is expected to be
more relevant in dense cultures of microalgae in which a
light gradient exists. Moreover, not all organisms respond
positively to increased mixing, particularly when fragile
cells such as mobile cells or filamentous organisms are
grown (Torzillo et al. 2003). The effect of mixing in
cultures of Scenedesmus obliquus on growth and N and P
removal was investigated by Martinez et al. (2000). They
concluded that stirring had a positive effect on biomass
productivity during the linear phase of growth, but that
increased stirring did not influence the removal of either P
or N. Moreover, increased mixing did not affect the
desorption of ammonia (loss by degassing; Martinez et al.
2000). In our experiments, mixing was found to be
beneficial for cultures of Chlorella, whilst the performance
in terms of biomass yields of unmixed co-cultures Chlorella
273
Fig. 4 PO43−–P removal by C. vulgaris, P. isothrix and co-cultures of
Planktothrix and Chlorella grown at 60 μmol photons m−2 s−1 in
shaken (a) and unshaken conditions (b). Chlorella (circle), co-culture
of Planktothrix and Chlorella (triangle), Planktothrix (inverted
triangle), control (without cells, diamond). Data on graph were fitted
with a one-phase exponential decay curve. Broken line indicates a
50% reduction in the initial phosphorus concentration
and Planktothrix was higher than that of mixed ones, a fact
that suggests different adaptation strategies for competing
in natural conditions.
We believe that the unshaken cultures, particularly of
Planktothrix as a single culture or in co-culture with
Chlorella, may have performed better because they were
not subjected to shear stress due to shaking and to the
negative effect of the continued resuspension of the
filaments throughout the water column. It is well known
that manipulation of the turbulent environment in an
appropriate manner can promote a species shift away from
cyanobacteria towards chlorophytes and diatoms (Steinberg
and Hartman 1988). A number of papers have reported a
reduction in the growth of Microcystis aeruginosa in
response to mixing (Reynolds et al. 1984; Visser et al.
1996). Initially, permanent de-stratification results in a
positive response with almost total removal of the cyano-
bacteria and nearly doubling in biomass of the chlorophytes
and diatoms in lakes (Steinberg and Gruhl 1992). Paerl and
Ustach (1982) suggested that buoyancy can be a part of an
ecological strategy aimed at making optimal use of
photosynthetically active radiation and atmospheric carbon
dioxide. From their investigations on Aphanizomenon
flos-aquae and Anabaena oscillatorioides, they concluded
that surface blooms would provide access to CO2 from the
air–water interface. Moreover, surface blooms markedly
274
influence the depth to which light penetrates the water
column, largely as a result of scattering by gas vacuoles
(Walsby 1994). Light scattering from the vertical increases
the path length of photons through horizontal water layers,
thereby enhancing the probability of photon capture within
the near surface layers. Those organisms which have taken
up preferential residence within the euphotic zone, such as
the cyanobacterium Planktothrix, have a greater probabil-
ity of intercepting light and thus have a distinct advantage
when competing with non-vacuolated species. On the
contrary, Chlorella, which is more resistant to shear stress
caused by mixing, performed better when the cultures
were shaken.
Nitrogen and phosphorus removal
Several authors have pointed out that the increase in pH due
to photosynthetic activity plays an important role in
wastewater bioremediation, promoting ammonia stripping
and P deposition as insoluble salts (Larsdotter 2006a). In
contrast, our study was aimed at minimising NH3 loss and
P precipitation and at incorporating them as much as
possible in the biomass. To this end, the pH of the cultures
was adjusted every day to the optimal value (pH 7.0).
Moreover, since ammonia is mostly present in the ionised
form below pH 8.0, the risk of ammonia of being toxic to
the cultures was prevented (Abeliovich and Azov 1976;
Park et al. 2010). According to Voltolina et al. (1998, 2005)
and Nuñez et al. (2001), only 25–33% of the initial NH4–N
content was recycled in the protein biomass by cultures of
S. obliquus, whilst the largest part was lost (presumably as
ammonia gas to the atmosphere) as a result of the combined
effect of the high pH values of the culture and of the
aeration. In our study, the amount of N recycled in the
biomass was considerably higher than that reported in the
literature, with up to 80% of which was removed. The
discrepancy between the recycled N and the N removed
may reasonably be ascribed to the loss of ammonia by
degassing as well as to the adsorption properties of the
cell walls of microorganisms (Volensky 2001; de Philippis
et al. 2007).
Nitrogen was never completely removed by the cultures,
although experiments were carried out for several days
under continuous illumination. This fact has been already
observed by Voltolina et al. (1998, 2005), Nuñez et al.
(2001) and by Tam and Wong (1996). The latter authors
found that the residual NH4–N in cultures of C. vulgaris
was <5% of the initial amount when the initial amount of
NH4–N was lower than 80 mg L−1. However, when the
initial amount was higher than 80 mg L−1, more than 50%
of the initial NH4–N still remained in the culture after
21 days, indicating that because of an excessive initial N
concentration in the cultures, it could not be totally taken
J Appl Phycol (2012) 24:267–276
up by the microalgal cells. Similar conclusions were also
drawn by Aslan and Kapdan (2006).
Phosphorus content in cells was not measured during the
experiments. However, since the pH of the cultures never
reached 8.5 units, i.e. the pH value at which the
precipitation of phosphate occurs, it is conceivable that
phosphates were mostly removed by active uptake rather
than being precipitated as insoluble phosphate salts.
However, biosorption phenomena by cell surface as well
as by the surface of the culture vessel cannot be ruled out.
Although the monoalgal cultures of Chlorella and the
co-culture of Chlorella and Planktothrix already removed
the P contained in the medium within 48–72 h from the
start of the experiment, their growth continued in a linear
manner. It is reasonable to suppose that the fast drop of P in
the medium could be due to luxury uptake, which has been
known to occur under conditions relevant for wastewater
treatment (Aitchison and Butt 1973). It has been found that
up to 3.16% of P can be stored in microalgal cells (Powell
et al. 2008). Similar conclusions were also reached by
Wang et al. (2010) when working with Chlorella sp. grown
in municipal wastewater and by Larsdotter (2006b).
Monster and Grobbelaar (1987) found that the P concen-
tration in microalgal cells depended on the supply concen-
tration and ranged approximately 0.1% of dry weight when
the concentration was 0.1 mg PL−1 to up to ten times higher
at a supply of 5 mg PL−1or more.
The use of microalgae-based wastewater treatment has
been shown to be effective in reducing the possibility of
eutrophication and other ecological damage, particularly in
tropical countries. In these countries, the stripping of
ammonia is further stimulated by higher temperature. Under
these conditions, nitrogen removal based on microalgae
may be expected to be highly efficient. However, the
concern with the increase in greenhouse gas may pose
severe limits in the use of ammonia stripping achieved
mainly by increased pH due to algal photosynthesis as a
way of reducing its content in the residual water.
Furthermore, ammonia in the atmosphere is converted
into N2O, which, over a 100-year period, has 298 times
more impact per unit weight than CO2. For these reasons,
considerable recycling of N in the biomass is desirable.
This can be achieved by controlling the pH of the culture
(Park et al. 2010). However, the additional cost of
supplying CO2 needs to be compensated by a higher
biomass yield, which should be usable for the extraction of
valuable products as well as for the production of energy
(e.g. biodiesel or biogas).
Various systems have been designed to provide the
mixing of cultures, the most common of which are
paddlewheels in shallow raceway ponds (Oswald 1989).
However, the economics of the process is still debatable
(Olguin 2003). Mixing and, particularly, biomass harvest-
J Appl Phycol (2012) 24:267–276
ing costs limit the economy of the process (Muñoz and
Guieysse 2006). Thus, the co-cultivation of two organisms,
the filamentous Planktothrix and the unicellular Chlorella,
which colonise two distinct layers of the wastewater
column in the absence of mixing, can be considered as
one of the possible options for removing P and N from
tropical wastewaters and may help reduce the costs of
wastewater treatment. This study has also demonstrated the
importance of pH control for reducing the emission of
ammonia into the atmosphere and the possibility of
achieving a greater recycling of nutrients into valuable
algal biomass.
Acknowledgements The work has been carried out within the
framework of the agreement between UCR and ISE-CNR. M. Silva is
indebted to the University of Costa Rica for enabling her to spend a
year visiting the ISE-CNR of Florence (Italy) for research purposes.
The authors would like to extend their thanks to Dr. Annarita Leva,
from CNR-IVALSA, for her statistical analysis.
References
Abeliovich A, Azof Y (1976) Toxicity of ammonia to algae in sewage
oxidation ponds. Appl Environ Microbiol 31:801–806
Aitchison EA, Butt VS (1973) The relationship between the synthesis
of inorganic polyphosphate and phosphate uptake by Chlorella
vulgaris. J Exp Bot 34:497–510
APHA, AWWA, WPCF (1992) Standard methods for the examination
of water and wastewater (1995), 18th edn. American Public
Health Association, Washington
Aslan S, Kapdan IK (2006) Batch kinetics of nitrogen and phosphorus
removal from synthetic wastewater by algae. Ecol Eng 28:64–70
Benemann JR, Weissman JC, Koopman BL, Oswald WJ (1977)
Energy production by microbial photosynthesis. Nature
268:19–23
Chevalier P, Proulx D, Lessard P, Vincent WF, de la Noüe J (2000)
Nitrogen and phosphorus removal by high latitude mat-forming
cyanobacteria for potential use in tertiary wastewater treatment. J
Appl Phycol 12:105–112
de-Bashan LE, Bashan Y (2004) Recent advances in removing
phosphorus from wastewater and its future use as fertiliser
(1997–2003). Water Res 38:4222–4246
de-Bashan LE, Trejo A, Huss VAR, Hernandez JP, Bashan Y (2007)
Chlorella sorokiniana UTEX 2805, a heat and intense, sunlight-
tolerant microalga with potential for removing ammonium from
wastewater. Bioresour Biotechnol 99:4980–4989
de la Noüe J, de Pauw N (1988) The potential of microalgal
biotechnology: a review of production and uses of microalgae.
Biotech Adv 6:725–770
de la Noüe J, Laliberté G, Proulx D (1992) Algae and wastewater. J
Appl Phycol 4:247–254
de la Noüe J, Lessard P, Proulx D (1993) Tertiary treatment of
secondarily treated urban wastewater by intensive culture of
Phormidium bohneri. Environ Technol 15:449–458
de Philippis R, Paperi R, Sili C (2007) Heavy metal sorption by
released polysaccharides and whole cultures of two
exoploysaccharide-producing cyanobacteria. J Appl Phycol
18:181–187
Driscoll CT, Lawrence GB, Bulger AJ, Butler TJ, Cronan CS, Eagar
C, Lambert KF, Likens GE, Stoddard JL, Weathers KC (2001)
275
Acidic deposition in the northeastern United States: source and
input, ecosystem effects, and management strategies. BioScience
51:180–198
Goldman JO (1979) Outdoor algal mass cultures. II. Photosynthetic
yield limitations. Water Res 13:119–136
Laliberté G, Proulx G, Pauw N, de la Noüe J (1994) Algal technology
in wastewater treatment. Ergeb Limnol 42:283–302
Larsdotter K (2006a) Wastewater treatment with microalgae: a
literature review. Vatten 62:31–38
Larsdotter K (2006b) Microalgae for phosphorus removal from
wastewater in a Nordic climate. Doctoral thesis, Royal Institute
of Technology, Stockholm, Sweden
Martinez ME, Sanchez S, Jiménez JM, El Yousfi F, Muñoz L (2000)
Nitrogen and phosphorus removal from urban wastewater by
microalga Scenedesmus obliquus. Biores Biotechol 73:263–272
Mostert ES, Grobbelaar JU (1987) The influence of nitrogen and
phosphorus on algal growth and quality in outdoor mass algal
cultures. Biomass 13:219–233
Muñoz R, Guiysse B (2006) Alga–bacterial processes for the
treatment of hazardous contaminants: a review. Water Res
40:2799–2815
Nuñez V, Voltolina D, Nieves M, Piña MA, Guerrero M (2001)
Nitrogen budget in Scenedesmus obliquus cultures with artificial
wastewater. Biores Technol 78:161–164
Ogbonna JC, Yoshizawa H, Tanaka H (2000) Treatment of high
strength organic wastewater by a mixed culture of photosynthetic
microorganisms. J Appl Phycol 12:277–284
Olguín EJ (2003) Phycoremediation: key issues for cost-effective
nutrient removal processes. Biotechnol Adv 22:81–91
Olguín EJ, Galicia S, Mercado G, Pérez T (2003) Annual productivity
of Spirulina (Arthrospira) and nutrient removal in a pig
wastewater recycling process under tropical conditions. J Appl
Phycol 15:249–257
Oswald WJ (1989) The role of microalgae in liquid waste treatment
and reclamation. In: Lembi CA, Waaland JR (eds) Algae and
human affairs. Cambridge University Press, Cambridge, pp 255–
281
Oswald WJ (2003) My sixty years in applied algology. J Appl Phycol
15:99–106
Oswald WJ, Golueke CG (1960) Biological transformation of solar
energy. Adv Appl Microbiol 2:223–262
Paerl HW, Ustach IF (1982) Blue-green algal scums: an explanation
for their occurrence during freshwater blooms. Limnol Oceanogr
27:212–217
Park JBK, Craggs RJ (2010) Wastewater treatment and algal
production in high rate algal ponds with carbon dioxide addition.
Water Sci Technol 61:633–639
Park J, Jin HF, Lim BR, Park KY, Lee K (2010) Ammonia removal
from anaerobic digestion effluent of livestock waste using green
alga Scenedesmus sp. Bioresour Technol 101:8649–8657
Park JBK, Craggs RJ, Shilton AN (2011) Wastewater treatment
high rate algal ponds for biofuel production. Biores Technol
102:35–42
Pinder RW, Gilliland AB, Dennis RL (2008) Environmental impact of
atmospheric NH3 emissions under present and future conditions
in the eastern United States. Geophys Res Lett 35:L12808
Pouliot Y, Buelna G, Racine C, de la Noüe (1989) Culture of
cyanobacteria for tertiary wastewater treatment and biomass
production. Biolog Waste 29:81–89
Powell N, Shilton AN, Pratt S, Chisti Y (2008) Factors influencing
luxury uptake of phosphorus by microalgae in waste stabilization
ponds. Environ Sci Technol 42:5958–5962
Rawat I, Kumar RR, Mutanda T, Bux F (2011) Dual role of
microalgae: phycoremediation of domestic wastewater and
biomass production for sustainable biofuel production. Appl
Energy. doi:10.1016/j.apenergy.2010.11.025
276
Reynolds CS, Wiseman SW, Clarke MJO (1984) Growth- and loss-
rate responses of phytoplankton to intermittent artificial mixing
and their potential application to the control of planktonic algal
biomass. J Appl Ecol 21:11–39
Richmond A (2003) Biological principles of mass cultivation. In:
Richmond A (ed) Handbook of microalgal culture: biotechnology
and applied phycology. Blackwell, Oxford, pp 125–177
Rippka R, Deruelles J, Waterbury J, Herdman M, Stanier R (1979)
Generic assignments, strain histories and properties of pure
cultures of cyanobacteria. J Gen Microbiol 111:1–61
Steinberg CEW, Gruhl E (1992) Physical measures to inhibit
planktonic cyanobacteria. In: Sutcliffe DW, Jones JG (eds)
Eutrophication: research and application to water supply.
Freshwater Biological Association, Cumbria, pp 163–184
Steinberg CEW, Hartmann HM (1988) Planktonic bloom-forming
cyanobacteria and eutrophication of lakes and rivers. Freshw Biol
20:279–287
Tam NFY, Wong YS (1996) Effect of ammonia concentrations on
growth of Chlorella vulgaris and nitrogen removal from media.
Biores Technol 57:45–50
Torzillo G, Pushparaj B, Masojidek J, Vonshak A (2003) Biological
constraints in algal biotechnology. Biotechnol Bioprocess Eng
8:338–348
J Appl Phycol (2012) 24:267–276
Valderrana LT, Del Campo CM, Rodroguez CM, de-Bashan LE,
Bashan Y (2002) Treatment of recalcitrant wastewater from
ethanol and citric acid production using the microalga Chlorella
vulgaris and the macrophyte Lemna minuscola. Water Res
36:4185–4192
Visser PM, Iberlings BW, Van Der Veer B, Koedood J, Mur LR
(1996) Artificial mixing prevents nuisance bloom of the
cyanobacterium Microcystis in Lake Nieuwe, the Netherlands.
Freshw Biol 36:435–450
Volensky B (2001) Detoxification of metal-bearing effluents: bio-
sorption for the next century. Hydrometallurgy 59:203–216
Voltolina D, Cordero B, Nieves M, Soto LP (1998) Growth of
Scenedesmus sp. in artificial wastewater. Biores Biotechnol
68:265–268
Voltolina D, Gomez-Villa H, Correa G (2005) Nitrogen removal and
recycling by Scenedesmus obliquus in semicontinuous cultures
using artificial wastewater and a simulated light and temperature
cycle. Biores Technol 96:359–362
Walsby AE (1994) Gas-vesicles. Microbiol Rev 58:94–144
Wang L, Min M, Li Y, Chen P, Cheng Y, Liu Y, Wang Y, Ruan R
(2010) Cultivation of green algae Chlorella sp. in different
wastewaters from municipal wastewater plant. Appl Biochem
Biotechnol 162:1174–1186