Biocatalysis and Agricultural Biotechnology 3 (2014) 126–131

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Biocatalysis and Agricultural Biotechnology

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Original Research Paper

Removal of nitrogen and phosphorus from wastewater using

microalgae free cells in bath culture system
Sara Rasoul-Amini a,b,c, Nima Montazeri-Najafabady a, Saeedeh Shaker b, Azam Safari b,
Aboozar Kazemi b, Pegah Mousavi b, Mohammad Ali Mobasher a, Younes Ghasemi a,b,n
a
Department of Pharmaceutical Biotechnology, school of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran
b
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran
c
Department of Medicinal Chemistry, school of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran

art ic l e i nf o a b s t r a c t

Article history: The effluents from wastewater contain nutrients (NH4þ , NO3 and PO4 3) which have been identified as
Received 21 May 2013 the main causes leading to eutrophication in natural waters. Therefore, the wastewater must receive
Received in revised form suitable treatment before being discharged into water bodies. Microalgae play an effective role during
30 August 2013
urban wastewater treatment. In this work five strains of microalgae growing as free-cells were used and
Accepted 3 September 2013
Available online 13 September 2013
compared to test their ability to remove nitrogen-nitrate (NO3 -N) and orthophosphate (PO34  -P)
in batch cultures of urban wastewater. The microalgae with the best cell growth configuration were
Keywords: selected, and introduced as a suitable strain for nutrient removal. Results indicate that Chlorella sp.
Chlorella sp. (YG01) showed a higher N uptake rate (84.11%) and Chlamydomonas sp. (YG04) and Chlamydomonas sp.
Chlamydomonas sp.
(YG05) showed a higher P uptake rate (100%) in urban wastewater than other species. Also during
Oocystis sp.
2 weeks of each experiment, most of the N and P removal was occurred at the first 4 days.
Microalgae
Nutrient removal & 2013 Elsevier Ltd. All rights reserved.
Wastewater treatment

1. Introduction to assimilate nutrients (Larsdotter, 2006). As reported in several

Because of the generation of a great volume of urban and
industrial wastewaters in industrialized countries, and also risk of
dumping these effluents into rivers, lakes or the sea, it is important
to treat wastewaters to reduce contaminants to environment
(Martinez et al., 2000). Inorganic substances (ammonium, nitrate
and phosphate) which encourage vegetal growth, contributing to the
eutrophication of the bodies of water containing the effluents, have
been received more attention in the waste water treatment
(Martinez et al., 2000). Therefore suitable treatment must be done
for urban wastewater before being discharged into water bodies.
Several types of processes are used for the removal of nutrients from
wastewater such as mechanical (influx, removal of large objects,
removal of sand and grit, primary sedimentation), chemical (disin-
fection) and biological (tricking bed filter, activated sludge) treat-
ments but these are costly and produce high sludge content. As an
alternative biological treatment, microalgae have been proposed to
remove nutrients from wastewater (Ruiz-Marin et al., 2010) and also
can be used for tertiary treatment of wastewater due to their capacity
n
Corresponding author at: Department of Pharmaceutical Biotechnology, school
of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz,
Iran. Tel.: þ 98 711 2424128; fax: þ 98 711 2426070.
E-mail address: ghasemiy@sums.ac.ir (Y. Ghasemi).
studies differences of nitrogen and phosphorus removal efficiencies
depend on the media composition and environmental conditions
such as the initial nutrient concentration, the light intensity, the
nitrogen/phosphorus ratio, the light/dark cycle and algae species
(Aslan and Kapdan, 2006).
Using microalgae in wastewater treatment has been studied
widely and indicated to have positive effect in nutrient removal
(Zamani et al., 2011). Microalgae are widespread in different
locations such as soil, air and fresh water (Rasoul-Amini et al.,
2011). The technology of using microalgae in wastewater treat-
ment is based on natural ecosystems; therefore there is no danger
for environment and even if the biomass produced is reused,
causes no secondary pollution (Zamani et al., 2011). Nitrogen
(N) and phosphorus (P) removal by microalgae in batch cultures
of wastewater can be done using immobilized and free-cells
techniques. In case the latter technique is used, harvesting free
cells in effluent is necessary to improve the quality of the treated
wastewater and avoid wash out of the biomass which potentially
can be used in food and pharmacy industries and/or as biogas
(Zamani et al., 2011).
In this research, we studied the kinetics of N and P elimination as
well as simultaneous growth of five microalgae strains in the effluent
from a secondary-sewage treatment, under constant conditions of
stirring and temperature. The aim of this study was to determine the

1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bcab.2013.09.003
 S. Rasoul-Amini et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 126–131
ability of different native strains of microalgae on the removal of N
and P by evaluating NO3 -N and PO3 4 -P loss and biomass
productivity.
2. Materials and methods
2.1. Microalgae isolation and cultivation
Five strains of isolated microalgae were used: two strains of
Chlamydomonas sp., two strains of Chlorella sp. and one strain of
Oocystis sp. Microalgae were isolated from agricultural soil samples
of paddy-fields of Fars province, Iran, from May to December 2011,
which were named YG01–YG05.
Soil samples were suspended in a specific volume of distilled
water. The supernatant was transferred to BG-11 solid culture medium
(Zamani et al., 2011), and Petri dishes were stored in a culture room
under constant illumination (4150 lx) with white fluorescent lamps at
2572 1C. After colonization, the isolation and purification were
performed using the plate agar method to obtain unialgal cultures
(Zamani et al., 2011). The microalgal cells were grown at room
temperature in liquid BG-11 medium with shaking at 130 rpm.
2.2. Identification and characterization
The taxonomic identification was done following the keys
of Desikachary (1959) and John et al. (2002). In order to determine
and confirm the species, the sequence of small subunit of 18S rRNA
was studied using molecular markers. Genomic DNA of microalgae
strains was prepared according to Rasoul-Amini et al. (2009). DNA
fragment of  700 was amplified from genomic DNA of microalgae
strains with polymerase chain reaction (PCR) by using universal
primers against the 18 S rRNA genes. The universal eukaryotic
primers 5′-GTCAGAGGTGAAATTCTTGGATTTA-3′ as forward primer
and 5′-AGGGCAGGGACGTAATCAACG-3′ as reverse primer, ampli-
fied a  700-bp region of the 18S rRNA genes (Zamani et al., 2011).
PCR amplifications were determined by 1% (w/v) agarose gel
electrophoresis in Tris/Borate/EDTA buffer. A single band of ampli-
fied DNA product of  700-bp was recorded. PCR products were
purified form agarose gel and used as templates in sequencing
reactions by CinnaGen company. 18S rRNA sequences were an
alyzed using the BLAST program, and published in the NCBI
databases under the specific accession numbers (Table 1).
The isolated microalgae were kept in the liquid nitrogen and
lyophilized in order to be added into Microalgal Culture Collection
(MCCS) of Shiraz University of Medical Science (Zamani et al.,
2011).
2.3. Preparation of municipal wastewater source and experimental
condition
Wastewater was collected from secondary effluent of Shiraz, Iran,
wastewater refinery. Filtered, and then sterilized by autoclave.
Approximately 3.5 ml from each free cells of microalgae were added
to 100 ml of autoclaved wastewater in 250 ml Erlenmeyer flasks for
Table 1
The published sequences of 18S rRNA of microalgae in the NCBI with their length
and accession numbers.
Microalgae Accession number Length (base pair)
Chlorella sp. (YG01) KC456059 613
Chlorella sp. (YG02) KC456060 620
Oocystis sp. (YG03) KC456061 336
Chlamydomonas sp.( YG04) KC456062 502
Chlamydomonas sp. (YG05) KC456063 560
127
each replication (pH¼9.0). The same volume of wastewater was
added to flasks with no cells of microalgae as the blank treatment.
Then, the flasks were placed in culture room at 2572 1C and
constant illumination (4150 lx). All treatments and blank wastewater
were conducted in three replicates (18 treatments in total).
The experiment lasted 14 days. Using microalgae growth curve,
achieving high biomass productivity in microalgae for 14 days was
shown. Using a sterile pipette, 2 ml of the wastewater was taken

every 4 days for PO3
4 -P and NO3 -N measurement according to the
Ultraviolet Spectrophotometric Screening method for nitrogen with
different concentration of KNO3 as standard and Ascorbic Acid
method for phosphorous with different concentration of K2HPO4 as
standard based on standard methods for the examination of water
and wastewater (Greenberg et al., 1998).
2.4. Determination of chlorophyll α and β carotene
In the first and last day of experiment 3 ml of the microalgae
suspension was taken for Chlorophyll α concentration determina-
tion according to the method described by Eijckelhoff and Dekker
(1997). Suspension of microalgae were centrifuged at 2500 rpm at
4 1C for 10 min and then rinsed with acetone (80%), then centri-
fuged again and analyzed using the mentioned method.
Beta-carotene concentration determined according to the
n-hexane method (Eijckelhoff and Dekker, 1997). In the first and
last day of experiment 1 ml of the microalgae suspension was
taken and centrifuged at 3000 rpm at 4 1C for 5 min and then
rinsed with ethanol/n-hexane (2/1) and distilled water respec-
tively and analyzed with n-hexane method (Eijckelhoff and
Dekker, 1997).
2.5. Statistical analyses
For all statistical analyses Paired-Samples T Test, SPSS Statistics
Software Version 19.0 was used. The mean, confidence interval,
P value and standard deviation values of the triplicates for each
treatment were calculated. The effects caused by urban waste-
water on the growth of several types of microalgae and nutrients
removal cultivated in free state were evaluated and statistical
significance of all treatments removal were evaluated.
3. Results and discussion
3.1. Identification of microalgae
The identified microalgal strains, based on chemotaxonomic and
18S rRNA data, cultivated in sterile BG-11 medium for the purpose of
this study, consisted of Chlorella sp. (YG01), Chlorella sp. (YG02),
Oocystis sp. (YG03), Chlamydomonas sp. (YG04) and Chlamydomonas
sp. (YG05). The lengths of the 18S rRNA region of five species of micro-
algae and their specific accession numbers are shown in Table 1.
3.2. Removal of nitrogen-nitrate under batch culture conditions
The concentration of NO3 -N and percent of changes during
different periods of time, and the whole period of the experiment
are given in Tables 2 and 3 (positive sign indicates the decrease
and negative sign indicates increase in NO3 -N concentration). The
changes in measured data are shown in Fig. 1.
The initial concentration of NO3 -N in the wastewater was
190.7 mg L  1 and decreased in almost all treatments to the minimum
value of 30.30 mg L  1 in Chlorella sp. (YG01) with the approximate
removal efficiency of 84.11% over 14 days (Po0.05). A continuous
increase and decrease in NO3 -N concentration occurred in blank and
samples, respectively, resulting in addition of removal of 84.11% from
128 S. Rasoul-Amini et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 126–131

Table 2
Nitrate concentration in the treatments during the experiment (mg L  1 d  1).

Treatment NO3 -N Concentration 7SD (mg L  1 d  1)

Day 1 Day 4 Day 7 Day 10 Day 14

Blank 190.7 70.12 193.25 7 0.08 193.25 7 0.08 194.75 7 0.05 195.64 70.06
Oocystis sp. (YG03) 190.7 70.12 120.28 7 0.09 43.93 7 0.01 36.727 0.02 31.81 70.18
Chlorella sp. (YG01) 190.7 70.12 124.237 0.16 111.747 0.22 46.107 0.12 30.30 70.03
Chlorella sp. (YG02) 190.7 70.12 92.65 7 0.25 70.807 00.4 65.717 0.21 60.58 70.20
Chlamydomanas sp.(YG04) 190.7 70.12 131.357 0.28 92.197 0.13 49.887 0.13 42.7770.08
Chlamydomanas sp.(YG05) 190.7 70.12 122.30 7 0.14 81.497 0.12 66.787 0.10 48.64 70.15

Table 3 was 94.77% after only 4 days (Po0.05). In all treatments orthopho-
Percent of changes in NO3  -N concentration in different periods of time.
sphate concentration decrease during the 14 days and depletion of
Treatment Percent of change NO3  -N (%) orthophosphate occurred over the whole period of the experiment.
Phosphorus removal in this study was obtained fast (4 days) for
Days 1–4 Days 1–7 Days 1–10 Days 1–14 Chlamydomonas sp. (YG04 and YG05), while the time required
for removing P in other studies have been longer for C. vulgaris. This
Blank  1.34  1.34  2.12  2.60
Oocystis sp. (YG03) 36.92 76.96 80.74 83.32 shorter period of treatment by free cells might be an indication that
Chlorella sp. (YG01) 34.85 41.40 75.82 84.11 the concentration of 19.11 mg L  1 of P in wastewater was enough to
Chlorella sp. (YG02) 51.41 62.87 65.54 68.23 provide adequate nutrients removal (Ruiz-Marin et al., 2010).
Chlamydomonas sp. (YG04) 31.12 51.66 73.84 77.57 According to Table 6 the specific removal rates of Chlamydo-
Chlamydomonas sp. (YG05) 35.87 57.27 57.27 74.49
monas sp. (YG04) were reported to be 1.95 mg P L  1 d  1.
All the differences are statically significant (Po 0.05).

250
3.4. Nitrogen and phosphorus removal rates

200 blanK In batch system, the initial substrate removal rates, Ri, are used
YG03 to determine the coefficients. The initial substrate removal rate is
Con.(mg L-1)

YG01 calculated as given in

150
YG02
S0 St
YG04 Ri ¼  ð1Þ
100 t 0 t t
YG05

50
0
1 4 7 10 14
Time(day)
Fig. 1. Nitrogen concentrations (mg L  1) of treatments in wastewater.
the best sample. In all treatments NO3 -N concentration decreases
during the 14 days and depletion of nitrogen-nitrate carried out over
the whole period of the experiment. Nitrogen removal in the present
study was achieved relatively fast, as mentioned in the Table 3, after
4 days 51.41% of nitrogen removal occurred by Chlorella sp. (YG02) and
Chlorella sp. (YG01) showed the best removal efficiency of 84.11% over
14 days while the time required to remove N in other studies have
been longer (8 days) for Chlorella vulgaris (Ruiz-Marin et al., 2010). As
the removal rate for defined concentration of NO3 -N is depicted in
Table 6, the removal rates of Chlorella sp. (YG01) were reported
11.46 mg N L  1 d  1 (Po0.05).
3.3. Removal of orthophosphate under batch culture conditions
The concentration of PO34  -P and percent of changes during
different periods of time, and the whole period of the experiment
are given in Tables 4 and 5 (positive sign indicates the decrease
and negative sign indicates increase in PO34  -P concentration). The
trend of changes in measured data is shown in Fig. 2.
The initial concentration of orthophosphate in the wastewater is
19.11 mg L  1 and it decreased in almost all treatments to the
minimum value in Chlamydomonas sp. (YG04 and YG05) with the
approximate removal efficiency of 100% over 14 days (Po0.05).
The removal of PO3 4 -P in the Chlamydomonas sp. (YG04 and YG05)
where Ri shows the substrate removal rate, S0 is the initial
substrate concentrations as NO3 -N or PO34  -P, St is the corre-
sponding substrate concentration at time “t” which is the time at
which concentration of the substance did not change significantly.
The slope of time versus effluent concentration at time “t” gives
the initial substrate removal rate. The specific rate of substrate
removal (Rxi) was determined by dividing the initial rates to
Chlorophyll α concentration at the start up of the experiments
(Aslan and Kapdan, 2006).
½Rxi ¼ Ri =ðChlorophyll αÞ0  ð2Þ
Wastewater contains large amount of materials like toxins, microbes,
etc. Basically, the main step to remove unwanted materials from
wastewater is processing. The type of processing depends on the
characteristics of wastewater. For nutrient removal from the waste-
water, single microalgae strain or compound of microalgae along
with microalgae growth-promoting bacteria are used (Sriram and
Seenivasan, 2012).
Using microalgae for wastewater treatment is the advanced
technology that based on natural ecosystems; so there is no danger
for environment and because of no secondary pollution, the biomass
produced is reused. Therefore, microalgae have great effect on
purifying the wastewater by producing oxygen and removing heavy
metals (Zamani et al., 2011).
Some techniques such as immobilized and free-cells were
mentioned to evaluate the ability of microalgae for nitrogen and
phosphorus removal in urban wastewater. In this study free-cell
technique has been done and the result has been considered.
An overall representation of the experimental data concentration
(mg L  1), removal phosphorous (mg L  1) and removal nitrogen
(mg L  1) in the growth experiments, in relation to time (day),
provides a clear and quick view of the trends of each culture.
 S. Rasoul-Amini et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 126–131 129

Table 4
Orthophosphate concentration in the treatments during the experiment (mg L  1 d  1).

Treatment PO34  -P Concentration 7 SD (mg L  1 d  1)

Day 1 Day 4 Day 7 Day 10 Day 14

Blank 19.117 0.03 17.707 0.04 16.247 0.05 12.34 70.02 10.447 0.03
Oocystis sp. (YG03) 19.117 0.03 14.94 7 0.03 6.727 0.04 4.58 70.05  0.52 7 0.01
Chlorella sp. (YG01) 19.117 0.03 6.577 0.14 6.217 0.04 5.55 70.01 3.377 0.02
Chlorella sp. (YG02) 19.117 0.03 5.59 7 0.08 5.047 0.00 3.96 70.02  0.477 0.00
Chlamydomanas sp.(YG04) 19.117 0.03 1.83 7 0.01 1.007 0.00  0.39 70.01  0.617 0.02
Chlamydomanas sp.(YG05) 19.117 0.03 1.007 0.01 0.58 7 0.01 0.0770.00  0.617 0.02

Table 5
Percent of changes in PO34  -P concentration of treatment in different periods of time.

Treatment Percent of changes PO43  -P (%)

Days 1–4 Days 1–7 Days 1–10 Days 1–14

Blank 7.38 15.02 35.43 45.37

Oocystis sp. (YG03) 21.82 64.83 76.03 99.01
Chlorella sp. (YG01) 65.62 67.50 70.96 82.36
Chlorella sp. (YG02) 70.75 73.63 79.28 99.00
Chlamydomonas sp. (YG04) 94.77 94.86 100.00 100.00
Chlamydomonas sp. (YG05) 94.77 96.96 99.63 100.00

All the differences are statically significant (Po 0.05).

Table 6
Nitrogen and phosphorus removal rate (mg L  1 d  1).

Treatment Ri (mg L  1 d  1) Rxi (mg L  1 d  1 chlorophyll α)

Ri (NO3 -N) Ri (PO34  -P) Rxi (NO3 -N) Rxi (PO34  -P)

Oocystis sp. (YG03) 11.35 1.40 20.27 2.5

Chlorella sp. (YG01) 11.46 1.12 23.87 2.44
Chlorella sp. (YG02) 9.29 1.39 17.66 2.24
Chlamydomonas sp. (YG04) 10.56 1.95 14.04 2.62
Chlamydomonas sp. (YG05) 10.15 1.90 13.68 2.52

blank The reported nitrogen and phosphorus removal efficiencies

25
YG03 show the basal depending on the media composition and envir-
YG01 onmental conditions such as the initial nutrient concentration, the
20
YG02 light intensity, the nitrogen/phosphorus ratio, and the light/dark
Con.(mg L-1)

YG04 cycle or algae species (Aslan and Kapdan, 2006).

15
YG05
10
5 (2003) culture of Botrycoccus braunii in pretreated piggery waste-
0 produced hydrocarbons. The phosphorus and nitrogen removal
1 4 7 10 14
Time(day)
Fig. 2. Phosphorous concentrations (mg L  1) of treatments in wastewater.
Wastewater samples were collected from secondary effluent of
the wastewater treatment, before chlorination; so autoclaving was
necessary to eliminate bacteria and pathogens (Hernandez et al.,
2006).
Municipal sewage contains various organic compounds such as
volatile acids, non-volatile soluble acids, fatty acids, amino acids
and carbohydrates, many of which are known to be used by algae
for mixotrophic and heterotrophic growth (Zhang et al., 2008).
Thus, for light-limited conditions, mixotrophic growth has a great
potential for the production of biomass and elimination of waste-
water nutrients.
Microalgae can be used in livestock wastewater treatment
processes, especially for reduction of excess nitrogen and phos-
phorus compound levels (An et al., 2003). As reported by An et al.
water reduced both inorganic nitrogen and phosphate levels, and
efficiencies achieved in this study were higher compared to some
of other studies previously published. For instance Dumas et al.
(1998) reported complete phosphorus removal by Phormidium
bohneri, the initial concentration was considerably lower
(0.05 mg PO34  -P L  1) compared to the concentrations used in
our study (Aslan and Kapdan, 2006).
3.5. Nitrogen removal
Nitrogen exists in many forms, and the most common nitrogen
compounds assimilated by microalgae are ammonium (NH4þ ) and
nitrate (NO–3) (Larsdotter, 2006).
The curve for removal of nitrogen over time reflects a specified
fall in the N concentration during the 14 days for the culture of
microalgae free-cells (Fig. 1). The 2.60% of increase in nitrogen
concentration in blank treatment during the experimental time is
130 S. Rasoul-Amini et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 126–131
due to the nitrification by bacteria, despite autoclaving. But in all
treatments NO3 -N concentration decrease during the 14 days and
depletion of nitrogen occurred over the whole period of the experi-
ment. Results indicate that Chlorella sp. (YG01) showed a higher
N uptake rate (84.11%) (Po0.05) in urban wastewater than other
species, and it was more effective in removing N within 14 days
when tested in bath cultures. In Chlorella sp. (YG01) treatment,
34.85% of decrease in nitrogen concentration occurred during the
first period (days 1–4), and the rest of the total decrease (41.40%)
occurred in 7 days (Po0.05). Then after 10 days 75.82% of nitrogen
was decreased and the last result after 14 days indicate 84.11%
reduction of nitrogen concentration in the treatment (Po0.05).
According to Table 3, there is a difference in N removal efficiency
of microalgae in the first period. Chlorella sp. (YG02) treatment,
indicate the maximum NO3 -N concentration decreased during the
first 4 days. But after 10 days Oocystis sp. treatment was the best one.
This difference is related to the microalgae ability to absorb and
decrease of nutrients from wastewater.
The free living cells of Chlorella sp. (YG01) in the bath culture
condition in this experiment had a higher nitrate removal rate
(84.11%) than free living cells of Chlorella sorokiniana (14.35%,
38.57% and 40.59%) under autotrophic, heterotrophic, and micro-
aerobic conditions, respectively (Liu et al., 2012).
3.6. Phosphorous removal
Another macro-nutrient that is essential for growth is phosphorus,
which is absorbed by algae as inorganic orthophosphate (PO3 4 ). The
uptake of orthophosphate is an active process that requires energy.
Phosphorus, which is stored in the cells are assimilated in the form of
polyphosphate granules by microalgae (Larsdotter, 2006).
As a result of the nature of the urban wastewater used, there
were variations in the concentration of PO34  -P in the waste-
water used for the experiment (Fig. 2). Therefore, removal
percentages were used to determine removal efficiency. The
removal of PO34  -P in the blank (no microalgae) was 45% and all
treatments with microalgae achieved higher removal than in
the blank. The decrease in phosphorous concentration in blank
treatment during the experimental time is due to the chemical
precipitation of calcium phosphate in terms of pH over eight
and high levels of calcium in the wastewater; according to the
formula:
5CaðOHÞ2 þ 3ðHn PO4 Þð3nÞ -Ca5ðOHÞðPO4Þ3↓ þ nH2 O þ ð9nÞOH
ð3Þ
Therefore nitrification by bacteria, despite of autoclaving, che-
mical precipitation orthophosphate and uptake it by bacteria as
a source of nutrition for growth and cell production causes a
reduction in the phosphorous in the blank treatment.
Experiments with free cells of microalgae showed that Chla-
mydomonas sp. (YG04, YG05) was more effective in removing
PO34  -P than other species after 14 day of treatment (Table 5).
The percentage of phosphorus removal for free-cells cultures of
Chlamydomonas sp. (YG04, YG05) showed a higher PO34  -P uptake
rate (100%) in urban wastewater than other species. For Chlamy-
domonas sp. (YG04, YG05) during the first period (days 1–4) in
average 94.77% of decrease in phosphorous concentration
occurred, but for Chlamydomonas sp. (YG04) after 7 days of total
experiment 100% of decrease have been showed (P o0.05). And
the results after 14 days indicate 100% reduction of PO34  -P
concentration in the treatment. According to Table 4, there is a
difference in P removal efficiency of microalgae treatments.
Xin et al. (2010) reported approximately the same result as this
study – nitrogen and phosphorus removal by Scenedesmus sp. LX1
in the growth medium with different nitrogen sources after 13
days of cultivation with nitrate, ammonium and urea as nitrogen
sources. Total nitrogen removal efficiencies were 90.4%, 31.1% and
87.8%; and total phosphorus removal efficiencies were 499%,
76.4% and 4 99%.
The phosphorus and nitrate-nitrogen removal efficiencies achieved
in this study were higher compared to some of other studies
previously published. For instance, an average of 72% nitrogen and
28% phosphorus removal by C. vulgaris containing diluted citric acid
and ethanol production obtained (Aslan and Kapdan, 2006).
Xin et al. (2010) reported that the order of specific growth rate of
the Scenedesmus sp. in different nitrogen sources was NH4þ -N4urea-
N4NO3 -N. This microalgae show well growth and remove both
nitrogen and phosphorus efficiently when use nitrate or urea as
nitrogen source (90% nitrogen and nearly 100% phosphorus were
removed).
The results of this study in terms of nitrogen removal for the
different concentrations are consistent with the other studies. For
instance, over 97% nitrogen and phosphorus removal was achieved
by Scenedesmus obliquus for the nutrient concentrations of 27.4
and 11.8 mg L  1 (Aslan and Kapdan, 2006).
Aslan and Kapdan (2006) obtained maximum removal 96% nitro-
gen and 87% phosphorous by Spirulina sp. in an outdoor raceway
treating 2% diluted anaerobic effluents from pig wastewater containing
almost the same amount of nitrogen and phosphorus as in the
experiment carried out by Martinez et al. (2000).
Nevertheless, there are some reports about efficient nutrient
removal at various concentrations. For example Aslan and Kapdan
(2006) reported about C. vulgaris and Scenedesmus dimorphus that
55% phosphorus removal from agro-industrial wastewater with
the total phosphorus concentration of 111 mg L  1 by 216 h batch
cultivation. However, our result was about 100% for PO34  -P
concentration.
An et al. (2003) reported about the effect of the initial phosphorus
and nitrogen concentration on nutrient removal efficiency of algae B.
braunii from secondarily treated piggery wastewater.
Growth and nitrate removal under phosphate excess in med-
ium containing different concentrations of nitrate are illustrated in
the report by Wang and Lan (2011).
Some reporters investigated a satisfactory nutrients removal
(about 99.9% for the nitrogen and phosphorus) and a specific
biomass productivity of 0.25 g/L d has been determined in the
indoor photo bioreactor. The results have been reached in the
outdoor photo bioreactor was less satisfactory because of ambient
condition instability and limiting nutrients concentration (Termini
et al., 2011).
Ruiz-Marin et al. (2010) reported NH4þ -N removal (100%) for
carrageenan-immobilized S. obliquus and also reported nitrogen
removal of 82% for alginate-immobilized C. vulgaris cultivated in
urban wastewater.
4. Conclusions
This experiment confirms that Chlorella sp. (YG01) and Chla-
mydomonas sp. (YG04, YG05) can be considered as efficient
nutrient removers from urban wastewater. They are capable of
completely depleting N and P correspondingly from wastewaters
containing high concentrations of nitrate and phosphate while
achieving high biomass productivity.
Further research is suggested on the utilization of locally
growing species for wastewater nutrient removal by immobilized
cells in different conditions such as bioreactor or semi-continuous
or bath culture. The diversity and seasonal dynamics of algal
species that naturally develop on wastewater ponds offer the
possibility of testing different species and selection of the best one.
 S. Rasoul-Amini et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 126–131
Acknowledgments
This work was supported by a grant from the Research Council
of Shiraz University of Medical Sciences, Shiraz University of
Medical Sciences, Shiraz, Iran.
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