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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
Department of Pharmaceutical Biotechnology, school of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran
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.
Nutrient removal & 2013 Elsevier Ltd. All rights reserved.
Wastewater treatment

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

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

1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
S. Rasoul-Amini et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 126–131 127

ability of different native strains of microalgae on the removal of N each replication (pH¼9.0). The same volume of wastewater was
and P by evaluating NO3 -N and PO3 4 -P loss and biomass added to flasks with no cells of microalgae as the blank treatment.
productivity. 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).
2. Materials and methods The experiment lasted 14 days. Using microalgae growth curve,
achieving high biomass productivity in microalgae for 14 days was
2.1. Microalgae isolation and cultivation 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
Five strains of isolated microalgae were used: two strains of Ultraviolet Spectrophotometric Screening method for nitrogen with
Chlamydomonas sp., two strains of Chlorella sp. and one strain of different concentration of KNO3 as standard and Ascorbic Acid
Oocystis sp. Microalgae were isolated from agricultural soil samples method for phosphorous with different concentration of K2HPO4 as
of paddy-fields of Fars province, Iran, from May to December 2011, standard based on standard methods for the examination of water
which were named YG01–YG05. and wastewater (Greenberg et al., 1998).
Soil samples were suspended in a specific volume of distilled
water. The supernatant was transferred to BG-11 solid culture medium 2.4. Determination of chlorophyll α and β carotene
(Zamani et al., 2011), and Petri dishes were stored in a culture room
under constant illumination (4150 lx) with white fluorescent lamps at In the first and last day of experiment 3 ml of the microalgae
2572 1C. After colonization, the isolation and purification were suspension was taken for Chlorophyll α concentration determina-
performed using the plate agar method to obtain unialgal cultures tion according to the method described by Eijckelhoff and Dekker
(Zamani et al., 2011). The microalgal cells were grown at room (1997). Suspension of microalgae were centrifuged at 2500 rpm at
temperature in liquid BG-11 medium with shaking at 130 rpm. 4 1C for 10 min and then rinsed with acetone (80%), then centri-
fuged again and analyzed using the mentioned method.
2.2. Identification and characterization Beta-carotene concentration determined according to the
n-hexane method (Eijckelhoff and Dekker, 1997). In the first and
The taxonomic identification was done following the keys last day of experiment 1 ml of the microalgae suspension was
of Desikachary (1959) and John et al. (2002). In order to determine taken and centrifuged at 3000 rpm at 4 1C for 5 min and then
and confirm the species, the sequence of small subunit of 18S rRNA rinsed with ethanol/n-hexane (2/1) and distilled water respec-
was studied using molecular markers. Genomic DNA of microalgae tively and analyzed with n-hexane method (Eijckelhoff and
strains was prepared according to Rasoul-Amini et al. (2009). DNA Dekker, 1997).
fragment of  700 was amplified from genomic DNA of microalgae
strains with polymerase chain reaction (PCR) by using universal 2.5. Statistical analyses
primers against the 18 S rRNA genes. The universal eukaryotic
primers 5′-GTCAGAGGTGAAATTCTTGGATTTA-3′ as forward primer For all statistical analyses Paired-Samples T Test, SPSS Statistics
and 5′-AGGGCAGGGACGTAATCAACG-3′ as reverse primer, ampli- Software Version 19.0 was used. The mean, confidence interval,
fied a  700-bp region of the 18S rRNA genes (Zamani et al., 2011). P value and standard deviation values of the triplicates for each
PCR amplifications were determined by 1% (w/v) agarose gel treatment were calculated. The effects caused by urban waste-
electrophoresis in Tris/Borate/EDTA buffer. A single band of ampli- water on the growth of several types of microalgae and nutrients
fied DNA product of  700-bp was recorded. PCR products were removal cultivated in free state were evaluated and statistical
purified form agarose gel and used as templates in sequencing significance of all treatments removal were evaluated.
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). 3. Results and discussion
The isolated microalgae were kept in the liquid nitrogen and
lyophilized in order to be added into Microalgal Culture Collection 3.1. Identification of microalgae
(MCCS) of Shiraz University of Medical Science (Zamani et al.,
2011). The identified microalgal strains, based on chemotaxonomic and
18S rRNA data, cultivated in sterile BG-11 medium for the purpose of
2.3. Preparation of municipal wastewater source and experimental this study, consisted of Chlorella sp. (YG01), Chlorella sp. (YG02),
condition Oocystis sp. (YG03), Chlamydomonas sp. (YG04) and Chlamydomonas
sp. (YG05). The lengths of the 18S rRNA region of five species of micro-
Wastewater was collected from secondary effluent of Shiraz, Iran, algae and their specific accession numbers are shown in Table 1.
wastewater refinery. Filtered, and then sterilized by autoclave.
Approximately 3.5 ml from each free cells of microalgae were added 3.2. Removal of nitrogen-nitrate under batch culture conditions
to 100 ml of autoclaved wastewater in 250 ml Erlenmeyer flasks for
The concentration of NO3 -N and percent of changes during
Table 1 different periods of time, and the whole period of the experiment
The published sequences of 18S rRNA of microalgae in the NCBI with their length are given in Tables 2 and 3 (positive sign indicates the decrease
and accession numbers. and negative sign indicates increase in NO3 -N concentration). The
changes in measured data are shown in Fig. 1.
Microalgae Accession number Length (base pair)
The initial concentration of NO3 -N in the wastewater was
Chlorella sp. (YG01) KC456059 613 190.7 mg L  1 and decreased in almost all treatments to the minimum
Chlorella sp. (YG02) KC456060 620 value of 30.30 mg L  1 in Chlorella sp. (YG01) with the approximate
Oocystis sp. (YG03) KC456061 336 removal efficiency of 84.11% over 14 days (Po0.05). A continuous
Chlamydomonas sp.( YG04) KC456062 502
Chlamydomonas sp. (YG05) KC456063 560
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).

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

S0 St
YG04 Ri ¼  ð1Þ
100 t 0 t t

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

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

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

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

Acknowledgments Liu, K., Li, J., Qiao, H., Lin, A., Wang, G., 2012. Immobilization of Chlorella sorokiniana
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