OPEN ACCESS
International Journal of Aquatic Science
ISSN: 2008-8019
Vol. 10, No. 1, 27-36, 2019
Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris and a
mixture of two algae in municipal wastewater treatment
Mohammad Hadi Abolhasani1*, Seyed Abbas Hoseini2, Rasool Ghorbani2 and Ordog Vince3
1) Department of Natural Resources, Faculty of Agriculture, Islamic Azad University, Khorasgan Isfahan Branch, Esfahan, Iran.
2) Department of Natural Resources, Agriculture and Natural Resources, Gorgan, Iran.
3) Faculty of Agricultural and Food Sciences, Institute Of Plant Biology, University of West Hungary.
Received: April-03-2018
Accepted: May-19-2018
Published: January-01-2019
Abstract: This experiment was conducted over a 15-day period to determine efficiency of two algae species, Scenedesmus obliquus,
Chlorella vulgaris, either singularly or in combination, in nutrient absorption from municipal wastewater. Algal cell counting was performed
daily, but water nitrate, nitrite, phosphate, ammonia, chlorophyll a, algal biomass, BOD and COD levels were determined every other day. At
the end of the experiment, water phosphate in treatments C0, M50 and S100 was zero. Also, water nitrate levels reached zero, only in
treatment M50 and all groups of treatment S. The maximum algal biomass was observed in treatment S0 (0.6 gL -1; p<0.05) at the end of the
experiment. Algal biomass of the treatment S groups was significantly lower than that of the other treatments (0.2 gL -1; p<0.05). The
maximum chlorophyll a with significant difference was observed in treatment S0. COD and BOD content showed constant trends among the
treatments. The lowest COD and BOD was observed in treatments S and C respectively (4.1 mgL -1; p<0.05; 1.3 mgL-1; p<0.05). Overall, Sc.
Obliquus was more efficient than Ch. vulgaris in biomass production, chlorophyll a concentration and nutrient absorption.
Keywords: Scenedesmus obliquus, Chlorella vulgaris, phosphate, municipal wastewater
Introduction
Huge amounts of anthropogenic municipal wastewater
are produced in the residential regions. Water and
detergents utilization in routine life, human activities
like construction and agriculture, discharge huge
amounts of nitrogen and phosphorus to the aquatic
ecosystems. Water nutrient enrichment and
eutrophication may affect the ecosystem structure and
function. Algae are photosynthetic organisms which
use sun light to convert carbon (carbon dioxide) into
other organic materials and release oxygen into
atmosphere (Huzisige and Ke, 1993; Gebre, 2017). It
is necessary to monitor aquatic ecosystems nitrogen
and phosphorus and prevent violation of the suitable
water criteria. Employ of micro-algae with high
wastewater treatment potential is a biological method
to suppress water nutrients such as nitrogen and
phosphorus (Zhang et al., 2017; De la Noiie and
Proulx, 1988). Wastewater treatment by micro-algae
has some advantages such as nitrogen and
phosphorus recycling, prevention of subsequent
pollution and biomass production which can be
applied for biofuel, cosmetic materials, hygienic
materials and food production (Voltolina, et al., 2004;
Yalcin , et al., 2006). Chlorella vulgaris is a green
single-cell alga with worldwide distribution; whereas,
Scenedesmus is a green non-motile alga (Lee, et al.,
() hadi.mha2001@yahoo.com
2001; Li, et al., 2010). Micro-algae are superior to
macro-algae and higher plants, because they grow
rapidly and are capable to adapt natural and artificial
conditions, which this adaptation is accompanied with
increased protein, carbohydrate and lipid synthesis
(Rasmussen, et al., 2008). Green algae have
outstanding ability to adapt in a variety of aquatic
ecosystem (Solisio, et al., 2008; Brodie et al., 2017).
Ch. vulgaris and Sc. obliquus are rapid growing
organisms, resistant to handling in rearing systems,
easy and inexpensive to produce, capable to tolerate
a wide range of temperature and pH; also they absorb
nutrients rapidly and even are able to absorb nitrogen
and phosphorus at very low ambient concentrations
(Li, et al., 2010; Abolhasani et al., 2018; Jung et al.,
2017). This experiment has been conducted on
different concentrations of municipal wastewater,
because water nutrient shortage may inhibit algae
growth and efficiency (Kong, et al., 2010).
The aim of this study was to determine growth
and efficiency of Ch. vulgaris and Sc. obliquus to
remove nutrients from different concentrations of
municipal wastewater, and to determine interaction of
the two algae when are mixed to treat the wastewater.
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
Materials and Methods
first sampling, t: time between the two sampling.
DT=log2e/SGR
The algae were cultured under static system condition
in liquid culture medium Z8 according to Lavens and
Sorgeloos (1996). 500-ml flasks filled with 250 ml
culture medium were used for this experiment, under
sterile condition and at 23°C±2 temperature.
Municipal wastewater of Gorgan City was obtained
from the main refinery at the final stage. The
wastewater was first filtered with filter paper and then
autoclaved to ensure lack of microbial load. This
experiment was conducted with 9 treatments and 4
replications, which include: Chlorella 0, 50, 100 (C0,
C50, C100), Scenedesmus 0, 50, 100 (S0, S50,
S100) and Chlorella-Scenedesmus mixture 0, 50, 100
(M0, M50, M100). Three concentrations of the
wastewater were used: 50% (1 part of sterile distilled
water was mixed with 2 parts of the wastewater),
100% (1 part of sterile distilled water was mixed with 1
part of the wastewater), and 0% (no dilution was
performed); initial nutrient concentrations of each
medium are presented in Table 1.
This experiment was conducted as a completely
randomized design using factorial method with two
factors: algae type (Chlorella, Scenedesmus and
mixed) and wastewater concentration (0, 50 and 100
percent). To monitor organic materials removal over
time, regression was used; whereas, to find
relationship among the measured parameters,
Pearson correlation test was used. The analyses were
performed in SPSS 17 software, and graphs were
drawn using Excel 2013.
Results
As phosphate and nitrate are the major nutrients in
wastewater, efficiency of the algae for these
compounds’ uptake was investigated during the
experiment. The effect of algae type, wastewater
concentration, time and interactions among them on
phosphate uptake was as follow (Tab. 2).
Tab. 1: Initial concentration of nutrients in each treatment
(mgL-1).
Compounds
Nitrite
Phosphate
Nitrate
Ammonia
0%
58.2
52.2
116.4
2.05
50%
38.9
24
107
1.55
Tab. 2: F-value for the effect of algae, wastewater
concentration and their interaction on phosphate removal.
100%
32.3
21.3
69.15
0.95
Time (Day)
2
4
6
8
10
12
14
Algae inoculation into each replication of the
treatments was conducted in a constant manner. For
this, 24000 cells were inoculated into each 500-l
flasks filled with 250 ml of autoclaved wastewater. In
the mixture treatment, 12000 cells from each algae
species were inoculated to the medium (Voltolina, et
al., 2004). Dry matter and chlorophyll a were
measured every other day according to Permila and
Rao (1997) and Aminot and Ray (1999). Cell counts
was daily made by hemocytometery method (Martınez
et al., 2000). Measurement of nutrients (phosphorus
and nitrate) was conducted every other day using
Wagtech kits (UK) and photometer apparatus. BOD5
was determined every other day according to
Gonzales et al. (1997). Specific growth rate (SGR)
and doubling time (DT) are two important factors
indicating reproduction rate of algae in culture
medium. SGR and DT were determined according to
the following formulas (Omori and Ikeda, 1984):
Con.
177.74**
231.65**
213.67**
174.57**
93.32**
55.59**
40.61**
Con.: Concentration ** P<0.01; * P<0.05
Alga×Con.
81.7*
598.7**
206.78**
9.43
89.3*
139.58**
121.1**
Algal type and wastewater concentration had
significant effect on phosphate uptake in the culture
medium during the experiment, but their interaction
was not significant at day 8.
According to the results (Tab. 3), phosphate and
nitrate concentrations significantly decreased during
the experiment. Wastewater phosphate was
completely depleted in M0, M50 and S100. The
maximum phosphate concentration was observed in
C50 group (2 mgL-1). S100 treatment had the lowest
phosphate during the experiment; but C0 treatment
had the highest phosphate during the first 8 days of
the experiment, and C50 treatment had the highest
phosphate in the last days of the experiment. On the
other hand, the mixed algae treatment had the higher
rate of phosphate uptake.
According to the Table 4, effect of the algae type,
wastewater concentration, time and their interactions
on nitrate uptake was as follow at different time.
SGR= (LnN2-LnN1)/t
N2: cell count in the second sampling; N1: cell count in the
Int. J. Aqu. Sci; 10 (1): 27-36, 2019
Alga
417.29**
915.41**
201.17**
171.38**
120.58**
74.17**
49.34**
2
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
Tab. 3: Phosphate concentration in different treatments during the experiment (mean ± SD).
Treatment
C0
C100
C50
M0
M100
M50
S0
S100
S50
0
50.4Aa
(±0.47)
21.3aC
(±0.37)
24aB
(±0.38)
50.4aA
(±0.47)
21.3aC
(±0.37)
24aB
(±0.38)
50.4aA
(±0.47)
21.3aC
(±0.37)
24aB
(±0.38)
2
40.4bA
(±0.6)
9Be
(±0.21)
22.5aC
(±0.42)
13.2bD
(±0.25)
7.2bF
(±0.17)
10.5bE
(±0.2)
30.3bB
(±0.5)
5.1bG
(±0.14)
22bC
(±0.42)
4
27cA
(±0.63)
8.1bE
(±0.21)
16bC
(±0.29)
10.8cD
(±0.23)
4.8cG
(±0.13)
6.5cF
(±0.15)
22.2cB
(±0.47)
2.8cH
(±0.1)
15cC
(±0.26)
6
24dA
(±0.68)
7.8bD
(±0.15)
10cC
(±0.1)
4.2dF
(±0.13)
3.1eG
(±0.1)
5.5cE
(±0.11)
12dB
(±0.14)
2.3dH
(±0.1)
9dC
(±0.12)
Day
8
13eA
(±0.67)
5.7cD
(±0.43)
8dC
(±0.5)
2.5eF
(±0.31)
2.4fF
(±0.3)
2.9dE
(±0.34)
10eB
(±0.41)
2dF
(±0.21)
8dC
(±0.5)
10
5.4fC
(±0.53)
4.8dD
(±0.41)
6eB
(±0.57)
2.2eF
(±0.34)
2.4fF
(±0.35)
1eG
(±0.28)
7.2fA
(±0.59)
1.12eG
(±0.2)
3.5eE
(±0.32)
12
2.4Gc
(±0.49)
4eA
(±0.54)
3fB
(±0.5)
1.8eD
(±0.24)
0.8gF
(±0.09)
0.8eF
(±0.09)
1gF
(±0.1)
0.65fG
(±0.07)
1.03fE
(±0.1)
14
0hF
1.6fB
(±0.12)
2fA
(±0.16)
1eC
(±0.08)
0.04hE
(±0.013)
0eF
0.8gD
(±0.01)
0gF
0.72fD
(±0.001)
Lowercase letters show significant difference within each row and uppercase letters within each column (α=0.05).
Tab. 4: F-values of effect of algae type, concentration and their interaction on nitrate uptake.
Time (Day)
2
4
6
8
10
12
14
Alga
354.5**
152.33**
320.89**
103.96**
92.69**
95.49**
76.87**
Con.
200.41**
161.98**
355.85**
89.87**
103.98**
90.44**
87.4**
Con.: Concentration ** P<0.01; * P<0.05
Alga×Con.
130.91**
91.23*
145.76**
85.88**
73.18
75.95**
34.21
the end of the experiment (18.9 mgL-1). M100
treatment had the lowest nitrate during the first 6 days
of the experiment; S50 treatment had the lowest value
from the day 6 until the end of the experiment; and the
highest nitrate was observed in C0 treatment
throughout the experiment (Tab. 5).
Similar to phosphate, nitrate uptake was
significantly affected by algal type and wastewater
concentration, interaction and wastewater concentration had no significant effects at days 10 and 14.
Nitrate was completely depleted at day 12 in S50
group and at the end of experiment in M50, S0 and
S100 groups, but C0 group had the highest nitrate at
Tab. 5: Nitrate values in different treatments during the experiment (mean ± SD).
Treatment
C0
C100
C50
M0
M100
M50
S0
S100
S50
0
116.4aA
(±2.76)
69.15aC
(±1.67)
107aB
(±1.98)
116.4aA
(±2.76)
69.15aC
(±1.67)
107aB
(±1.98)
116.4aA
(±2.76)
69.15aC
(±1.67)
107aB
(±1.98)
2
83.52bA
(±1.12)
44.52bD
(±1.01)
51.15bB
(±1.08)
48.98bC
(±1.04)
11.16bF
(±0.7)
33.45bE
(±0.9)
53.3bB
(±1.07)
18.06bF
(±1)
49.85bC
(±1.03)
4
79.74cA
(±2.1)
35cD
(±1.4)
45.85cC
(±1.7)
46.5cB
(±1.7)
9.96cG
(±0.9)
14.85cF
(±1.12)
29.76cE
(±1.37)
12.8cG
(±1.1)
10.65cG
(±1.01)
6
70.74dA
(±2.72)
28.44dC
(±1.31)
18.6dE
(±1.21)
34.08dB
(±1.52)
9.18cG
(±0.91)
12.85dF
(±1.1)
24.48dD
(±1.23)
5.61dH
(±0.8)
9.98cG
(±0.93)
Day
8
59.4eA
(±2.12)
26.9dB
(±1.73)
10.72eE
(±1.34)
13.02eD
(±1.38)
7.2dG
(±1.01)
8.85eF
(±1.08)
21.24eC
(±1.43)
3.72eH
(±0.9)
5.4dH
(±0.98)
10
36.7fA
(±1.98)
21.66eB
(±1.25)
7.55fC
(±1.09)
4.7fD
(±1.01)
4.65eD
(±1.01)
1.06fF
(±0.9)
3.1fE
(±0.98)
2.3fF
(±0.94)
1.51eF
(±0.67)
12
30.48gA
(±1.45)
16.89fB
(±0.97)
3.53gC
(±0.12)
1.94gD
(±0.1)
1.42fD
(±0.09)
0.18fE
(±0.01)
0.58gE
(±0.03)
1.15fE
(±0.08)
14
18.9hA
(±1.13)
5.64gB
(±0.9)
0.65hC
(±0.09)
0.36gC
(±0.05)
0.27fC
(±0.01)
0fE
0fC
Lowercase letters show significant difference within each row and uppercase letters within each column (α=0.05).
Int. J. Aqu. Sci; 10 (1): 27-36, 2019
2
0fC
0gC
0fC
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
Figure 1 shows that treatments C50 and C100
(89% and 92% respectively) removed lower
phosphate percentage than the other treatments; also,
treatments C0 and C100 (83% and 91%) removed
lower nitrate percentage than that of the other
treatments. C0 group removed significantly lower
nitrate percentage than C100; both groups nitrate
removal percentages were significantly lower than that
of the other treatments. Treatments C0, M50 and
S100 had significantly higher phosphate removal
percentages than that of the other treatments. Overall,
Ch. vulgaris efficiency in nitrate and phosphate
removal was lower than the other treatments
(P<0.05).
Fig. 3: Phosphate removal trend in Chlorella treatment.
Phosphate removal was faster in concentration 0,
compared to the other concentrations, and increase in
concentration slowed down the phosphate removal
(Fig. 4).
Fig. 1: Percentage of nitrate and phosphate removal in
different treatments at the end of the experiment. Different
letters show significant difference among the treatments
(α=0.05).
Fig. 4: Phosphate removal pattern in Scenedesmus
treatment.
With regard to Figure 2, the treatments efficacy in
phosphate uptake was relatively similar, when mixed
algae are used. Among the Chlorella treatments, C0
group was significantly more efficacious to remove
phosphate from culture media (Fig. 3). Phosphate
uptake in Scenedesmus groups was very fast, as the
phosphate of medium approximately reached zero at
the day 10.
Nitrate removal from the media was very fast.
Nitrate removal was faster in concentration 0,
compared to the other concentrations, and increase in
concentration slowed down nitrate removal (Fig. 5).
Nitrate uptake in Chlorella treatments was lower than
the other treatments, and the lowest level was
observed in C0 group (Fig. 6). Nitrate uptake in
Scenedesmus was very fast, and the fastest uptake
was observed in S0 group (Fig. 7).
Algae biomass is directly related to medium
nutrient concentration. As shown in Table 6, algae
biomass in Chlorella treatments showed no significant
change during the experiment; whereas, in
Scenedesmus and mixed treatments algae biomass
significantly changed during the experiment. The
highest biomass was observed in S0 treatment (0.6
gL-1); whereas, the lowest biomass was observed in
the Chlorella treatment (C0, C50, C100; 0.2 g L-1).
Biomass in C100 treatment was 0.1 g L-1 during the
first 8-day of the experiment.
Fig. 2: Phosphate removal trend in mixed algae treatment.
Int. J. Aqu. Sci; 10 (1): 27-36, 2019
2
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
Fig. 5: Nitrate removal pattern in mixed algae treatment.
Fig. 6: Nitrate removal pattern in Chlorella treatment.
Fig. 7: Nitrate removal pattern in Scenedesmus treatment.
Tab. 6: Biomass in different treatments during the experiment (mean ± SD).
Treatment
C0
C100
C50
M0
M100
M50
S0
S100
S50
2
0bB
(±0.01)
0cB
(±0.01)
0dB
(±0.01)
0.1dA
(±0.01)
0.1bA
(±0.01)
0.1cA
(±0.01)
0.1dA
(±0.01)
0.1dA
(±0.01)
0.1cA
(±0.01)
4
0.1aA
(±0.01)
0cC
(±0.01)
0.1cB
(±0.01)
0.1dB
(±0.01)
0.1bB
(±0.01)
0.1cB
(±0.01)
0.2cA
(±0.02)
0.1dB
(±0.01)
0.1cB
(±0.01)
Day
8
0.2aB
(±0.01)
0.1bC
(±0.01)
0.2bB
(±0.01)
0.2cB
(±0.01)
0.3aA
(±0.02)
0.3bA
(±0.02)
0.2cB
(±0.01)
0.3bA
(±0.02)
0.2bB
(±0.01)
6
0.2aB
(±0.02)
0.1bC
(±0.01)
0.2bB
(±0.02)
0.2cB
(±0.02)
0.3aA
(±0.02)
0.3bA
(±0.02)
0.2cB
(±0.02)
0.2cB
(±0.02)
0.2bB
(±0.02)
10
0.2aD
(±0.01)
0.1bE
(±0.01)
0.3aC
(±0.02)
0.3bC
(±0.02)
0.3aC
(±0.02)
0.4aB
(±0.03)
0.5bA
(±0.03)
0.3bC
(±0.02)
0.2bC
(±0.01)
12
0.2aE
(±0.01)
0.2aE
(±0.01)
0.2bE
(±0.01)
0.4aC
(±0.03)
0.3aD
(±0.02)
0.4aC
(±0.03)
0.6aA
(±0.04)
0.5aB
(±0.04)
0.4aC
(±0.03)
14
0.2aE
(±0.01)
0.2aE
(±0.01)
0.2bE
(±0.01)
0.4aC
(±0.02)
0.3aD
(±0.02)
0.4aC
(±0.02)
0.6aA
(±0.03)
0.5aB
(±0.03)
0.4aC
(±0.02)
Lowercase letters show significant difference within each row and uppercase letters within each column
(α=0.05).
Overall, the Scenedesmus treatments had
signific-antly higher biomass compared to the other
treatments, at the end of the experiment (P<0.05).
However, the Chlorella treatments had significantly
lower biomass from the first day of the experiment.
The mixed treatments’ biomass was intermediate with
the highest biomass in M0 and M50, and the lowest in
Int. J. Aqu. Sci; 10 (1): 27-36, 2019
M100.
Chlorophyll a values are shown in Table 7.
Chlorophyll of the Scenedesmus treatments was
significantly higher than the other treatments at the
last days (14th) of the experiment. Also, chlorophyll
concentration in the treatments M was higher than
those of the C group. Overall, the highest chlorophyll
2
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
value was observed at the day 10 in M50 treatment
(4.97 mgL-1), but the lowest value was related to C100
at the day 2 (2.91 mgL-1). Mean chlorophyll value at
the day 10 was higher than the other sampling days in
all groups. The lowest chlorophyll values were
observed in the Chlorella treatments at all the
sampling days. Among the Chlorella treatments, C100
treatment had the lowest chlorophyll value. Overall,
S0 treatment had significantly higher chlorophyll at the
end of the experiment (P<0.05); whereas, C100
treatment had the lowest chlorophyll. During the
experiment, M50 treatment had the highest value
(P<0.05). Chlorophyll value in M50 treatment is
correlated to algae cell graph, which shows the
highest number a t the day 8, and increase in algae
cell number has resulted in biomass augment in M50
treatment.
Tab. 7: Chlorophyll a values in different treatments during the experiment (mean ± SD).
Treatment
C0
C100
C50
M0
M100
M50
S0
S100
S50
2
1.6gE
(±0.67)
1.5gE
(±0.62)
1.55fE
(±0.63)
1.7eC
(±0.71)
1.88fB
(±0.74)
1.77cC
(±0.72)
2.22eA
(±0.81)
1.84fB
(±0.71)
1.9fB
(±0.82)
4
3.5bD
(±0.5)
2.91bE
(±0.45)
3.12bD
(±0.46)
4.25aB
(±0.65)
3.94bC
(±0.61)
4.85aA
(±0.72)
4.4bB
(±0.67)
3.88bC
(±0.58)
4.41bB
(±0.67)
Day
8
3.1cC
(±0.49)
2.96bC
(±0.47)
3.04bC
(±0.51)
3.64bB
(±0.76)
3.71cB
(±0.78)
3.68bB
(±0.77)
4.4bA
(±0.85)
3.71cB
(±0.78)
3.77cB
(±0.79)
6
2.83eC
(±0.58)
2.89cC
(±0.6)
2.98cC
(±0.61)
3.57bB
(±0.76)
3.02dB
(±0.65)
3.27cB
(±0.72)
4.33bA
(±0.88)
3.23dB
(±0.64)
3.43cB
(±0.7)
10
3.9aC
(±0.81)
3.16aD
(±0.61)
3.27aD
(±0.65)
3.43cC
(±0.67)
4aB
(±0.88)
4.97aA
(±0.91)
4.87aA
(±0.95)
4.28aB
(±0.9)
4.64aB
(±0.93)
12
2.91dD
(±0.54)
2.46dD
(±0.42)
2.65dD
(±0.46)
3.42cB
(±0.78)
2.98dD
(±0.57)
3.53bB
(±0.81)
3.78cA
(±0.83)
3.3dC
(±0.71)
3.1dC
(±0.68)
14
2.65fB
(±0.55)
2.13eB
(±0.45)
2.2eB
(±0.47)
2.83dB
(±0.59)
2.23eB
(±0.48)
2.45cB
(±0.51)
3.17dA
(±0.64)
2.37eB
(±0.49)
2.27eB
(±0.49)
Lowercase letters show significant difference within each row and uppercase letterswithin each column
(α=0.05).
In the first days of the experiment (until day 6),
cell count in Chlorella group (C0 and C50) was higher
than the other groups. In the middle of the experiment
(until day 10) M treatments (M0 and M50) had the
highest cell count. At the end of the experiment (the
day 10), S treatments (S0, S100 and S50) had the
highest cell count. During the whole period of the
experiment, the highest cell count was observed at
the day 8 in M50 treatment (in M group, total number
of Chlorella and Scenedsmus cells was counted).
Among the Scenedesmus treatments, S0 treatment
had the highest cell count at the day 12, whereas,
among the Chlorella treatments, C) treatment had the
highest cell count. Overall, the Scenedesmus
treatments showed an increasing pattern, but
Chlorella treatments showed increasing pattern in the
first days of the experiment and decreasing pattern in
the last days of the experiment (Tab. 8). Totally, cell
count of M50 treatment and that of S50 treatment
were highest at the day 8 and the end of the
experiment among the treatments respectively
Int. J. Aqu. Sci; 10 (1): 27-36, 2019
(P<0.05).
BOD5 showed decreasing pattern during the study
period (Tab. 9). During the experiment, Chlorella
group had higher BOD than the other treatments;
among which C50 treatment had higher BOD than the
other treatments. Scenedesmus treatments had the
lowest BOD among the treatments; M treatments had
intermediate BOD, which was similar to that of the
Chlorella group in the first days of the experiment.
This pattern continued until the last day of the
experiment, but it was significantly lower than that of
the Chlorella treatment. There was no significant
difference in BOD among the Scenedesmus
treatments, but they had lower BOD than the other
treatments (P>0.05). The Chlorella treatment had
similar BOD, which was significantly higher than those
of S and M treatments (P<0.05).
COD was similar to BOD5 between groups during
the study period (Tab. 10). SGR of the Chlorella group
was significantly lower than the other treatment.
Scenedesmus treatments had the highest SGR,
3
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
Tab. 8: Algae cell number (x0.00001) in different treatments during the experiment (mean ± SD).
Treatment
C0
C100
C50
M0
M100
M50
S0
S100
S50
2
1.1bA
(±0.47)
1.13aA
(±0.48)
0.87cB
(±0.31)
1.12gA
(±0.47)
0.98cB
(±0.35)
1.04fD
(±0.4)
0.59eC
(±0.21)
0.46eC
(±0.16)
0.36gC
(±0.12)
4
12aA
(±1.12)
1.07bD
(±0.56)
9.78bB
(±1.06)
6.68cC
(±0.94)
2.29aD
(±0.71)
3.86cD
(±0.82)
2.32dD
(±0.72)
1.11dE
(±0.58)
1.17fE
(±0.6)
6
14.24bC
(±1.76)
0.84bA
(±0.12)
11.74aA
(±1.32)
7.72bB
(±1.21)
1.22bC
(±0.23)
10.49aA
(±1.25)
2.93dD
(±0.29)
1.32cE
(±0.24)
1.7eE
(±0.35)
Day
8
1.24bD
(±0.21)
1.12aD
(±0.14)
0.67cE
(±0.01)
14.49aA
(±1.22)
1.57bD
(±0.24)
32.74dB
(±2.35)
3.97cB
(±0.89)
2.2bC
(±0.65)
2.87dC
(±0.71)
10
0.98cF
(±0.12)
0.9bF
(±0.1)
0.53cF
(±0.09)
12.66eB
(±2.13)
1.59bE
(±0.23)
27.1bD
(±2.98)
10bC
(±1.75)
2.3bD
(±0.46)
4.33cD
(±0.78)
12
1.4bD
(±0.61)
0.22cD
(±0.07)
0.63cD
(±0.08)
2.18dC
(±0.71)
1.21bC
(±0.53)
2.72dC
(±0.75)
12.12aC
(±1.43)
2.4bC
(±0.75)
4.76bB
(±0.89)
14
0.72cC
(±0.11)
0.25cC
(±0.09)
0.56cC
(±0.1)
1.57fB
(±0.43)
2.17aB
(±0.67)
2eB
(±0.63)
4.49cA
(±1.1)
2.83aB
(±0.93)
5.87aA
(±1.23)
Lowercase letters show significant difference within each row and uppercase letters 0 within each column
(α=0.05).
Tab. 9: BOD5 value in different treatments during the experiment (mean ± SD).
Treatment
C0
C100
C50
M0
M100
M50
S0
S100
S50
2
4.86aA
(±0.98)
2.74aC
(±0.67)
3.91aB
(±0.89)
3.72aB
(±0.85)
2.56aC
(±0.57)
2.26aC
(±0.43)
1.59aD
(±0.34)
1.56aD
(±0.32)
2.1aD
(±0.4)
4
2.77bA
(±0.86)
2.24bB
(±0.8)
3bA
(±0.88)
2.9bA
(±0.89)
2.2bB
(±0.79)
2bC
(±0.79)
1.2bD
(±0.61)
1.2bD
(±0.61)
1.92bD
(±0.74)
Day
8
1.86cB
(±0.85)
1.94cA
(±0.87)
2.42dA
(±0.98)
2.32cA
(±0.96)
1.96cA
(±0.88)
1.6cB
(±0.7)
1cC
(±0.13)
1.08cC
(±0.14)
1.72cB
(±0.74)
6
1.94cA
(±0.92)
1.66cB
(±0.74)
2.3cA
(±0.99)
2.06cA
(±0.97)
1.75cB
(±0.78)
1.5cB
(±0.7)
0.91cC
(±0.09)
1cC
(±0.13)
1.5cC
(±0.56)
10
1.64dB
(±0.71)
2cA
(±0.97)
2.18eA
(±0.99)
2dA
(±0.97)
1.82cB
(±0.96)
1.4dB
(±0.67)
0.9cC
(±0.23)
0.98cC
(±0.26)
1.6cC
(±0.69)
12
1.68dB
(±0.71)
1.78cB
(±0.74)
1.8fB
(±0.8)
2dA
(±0.84)
1.62dB
(±0.68)
1.2eC
(±0.5)
0.8dD
(±0.24)
0.94cD
(±0.31)
1.2dD
(±0.5)
14
1.52eA
(±0.71)
1.56cA
(±0.72)
1.48gA
(±0.65)
1.4eB
(±0.61)
1.36dB
(±0.54)
1fC
(±0.31)
0.7dC
(±0.13)
0.72dC
(±0.14)
0.78eC
(±0.15)
Lowercase letters show significant difference within each row and uppercase letters within each column
(α=0.05).
Tab. 10: COD value in different treatments during the experiment (mean ± SD).
Treatment
C0
C100
C50
M0
M100
M50
Int. J. Aqu. Sci; 10 (1): 27-36, 2019
2
24.31aA
(±1.56)
13.72aC
(±1.1)
19.56aB
(±1.41)
18.6aB
(±1.37)
12.8aC
(±1)
11.3aC
(±0.98)
4
13.88bA
(±1.54)
11.2aB
(±1.34)
15bA
(±1.64)
14.5bA
(±1.58)
11aB
(±1.32)
10aB
(±1.23)
6
11.64bA
(±1.56)
10bB
(±1.2)
13.8bA
(±1.78)
12.4cA
(±1.67)
10.5aB
(±1.23)
9bB
(±1.1)
2
Day
8
9.3bB
(±1.23)
9.7bB
(±1.27)
12.1bA
(±1.87)
11.6cA
(±1.76)
9.8bB
(±1.28)
8cC
(±1.11)
10
8.2cB
(±1.12)
10bA
(±1.25)
10.9cA
(±1.29)
10cA
(±1.25)
9.1bA
(±1.17)
7dB
(±1.03)
12
8.4cB
(±1.24)
8.9cA
(±1.28)
9cA
(±1.29)
10cA
(±1.34)
8.1cB
(±1.22)
6eC
(±1.08)
14
7.6cA
(±1.34)
7.8cA
(±1.37)
7.4dA
(±1.3)
7dA
(±1.27)
6.8dB
(±1.25)
5fB
(±1.1)
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
Tab. 10: continued.
Treatment
S0
S100
S50
2
7.95aD
(±0.9)
7.8aD
(±0.9)
10.5aC
(±0.99)
4
6bC
(±1.1)
6bC
(±1.1)
9.6aB
(±1.14)
Day
8
5bD
(±0.98)
5.4cD
(±1.09)
8.6bC
(±1.14)
6
5.5bC
(±0.76)
6bC
(±0.98)
9bB
(±1.1)
10
4.5cC
(±0.87)
4.9cC
(±0.9)
8bB
(±1.08)
12
4cD
(±0.9)
4.7cD
(±1.01)
6cC
(±1.08)
14
3.5dC
(±1.06)
3.6dC
(±1.07)
3.9dC
(±1.09)
Lowercase letters show significant difference within each row and uppercase letters within each column
(α=0.05).
however, M100 treatment had the highest SGR (2.98).
DT showed inverse trend compared to SGR as the
highest DT was related to Chlorella treatments and
C50 (0.7) and the lowest DT was observed in
Scenedesmus group (Fig. 8).
highest nitrate was related to C0 treatment suggesting
that Chlorella is more efficient in phosphate removal
and Scenedesmus is more efficient in both nitrate and
phosphate removal. However, M50 was the only
treatment that completely depleted the medium
phosphate and nitrate suggesting that the two algae
mixture was more efficient among the treatments. Lee
and Lee (2001) reported that phosphate removal rate
by Chlorella kessleri is 28% with initial phosphate
concentration of 10 mgL-1 in culture medium.
However, Mallick et al.
(2002) reported that
Phormidium bonheri was capable to completely
remove phosphate and nitrate with initial
concentrations of 30 and 42 mgL-1 in culture medium.
Similar to the our results, it was found that Sc.
obliquus is capable to remove 97% of nitrogen and
phosphorus within 14 days with initial concentration of
27.4 and 11.8 mgL-1 (Martinez et al., 2000).
Scenedesmus obliquus is able to remove 99.1% of
wastewater nitrogen (NH4-N) within 12 days with initial
concentration of 3.8 mgL-1 (Yang et al., 2011). In our
study, Chlorella reproduced with a high rate in the first
days of the experiment, but it showed declining trend
after the day 8 suggesting lower tolerance of Chlorella
to culture medium conditions compared to the two
other groups. In the mixed algae group, increasing
pattern started at the day 4 continued until the day
10th, but showed decreasing pattern in the last days.
However, in Scenedesmus group an increasing
pattern was observed during the experiment
suggesting its high resistance to the culture medium
conditions. The highest chlorophyll value was
observed in S0 treatment; Scenedesmus and
Chlorella had significantly the highest and the lowest
chlorophyll values among all treatments had. M50
treatment had the highest chlorophyll due to
synergistic effect. This treatment had the highest cell
count at the day 8th suggesting correlation between
cell count and chlorophyll content. According to Fig. 4
and 5, under constant conditions, each Chlorella cell
has higher chlorophyll value than each Scenedesmus
Fig. 8: SGR and DT in different treatments during the
experiment. Different letters show significant difference
during the experiment (α=0.05).
Overall, S50, M100 and S100 treatments had
significantly the highest DT and the lowest SGR
during the experiment (P>0.05). However, the lowest
DT was related to C50 and C100 (P<0.05). M0 and
M50 treatments’ DT was between the Chlorella and
Scenedesmus groups, but had significantly higher DT
and lower SGR than the Chlorella group. C0 treatment
was similar to M0 and M50, but the other Chlorella
treatments were significantly lower than the other
treatments.
Discussion
The results showed that Chlorella, Scenedesmus and
mixed treatments had the highest phosphate uptake
at 0%, 100% and 50% wastewater dilution,
respectively, which phosphate was completely
depleted. Phosphate uptake rate was higher in the
first days compared to the last days of the experiment.
All Scenedesmus groups and M50 treatment
completely depleted the medium nitrate, but the
Int. J. Aqu. Sci; 10 (1): 27-36, 2019
3
Abolhasani et al. (2019) Evaluation the efficiency of Scenedesmus obliquus, Chlorella vulgaris …
cell. The highest and the lowest algal biomass was
found in Scenedesmus and Chlorella groups,
respectively. The mixed treatment, although was close
to Scenedesmus group, had significantly lower
biomass than this group. It is suggested that culture
medium nitrogen had more important role than
phosphorus in algae biomass production; this explain
weak biomass production in C0 treatment despite
complete phosphate uptake. Ruiz-Marin et al. (2010)
found that in wastewater as culture medium, growth
rate of Ch. vulgaris (8 h) is higher than Sc. obliquus
(20 h), which is in agreement with the present results.
Also, they reported that Sc. obliquus is more
resistance to various medium conditions than Ch.
vulgaris, and S. obliquus had significantly higher
chlorophyll-a at the end of the experiment (0.54 vs.
0.22 mgL-1). Tam and Wong (2000) reported than high
ammonium uptake by C. vulgaris is due to reaction
between carboxyl group in the outer membrane and
ammonium molecules. Chlorophyll and biomass of S.
obliquus and C. vulgaris was found to be related
energy intake (light) and self-shading in culture
medium. Ruiz-Marin et al. (2010) reported that
chlorophyll and biomass of S. obliquus was higher
than C. vulgaris (0.56 vs. 0.31 g L-1). Kaewsarn (2002)
showed that C. vulgaris has low efficiency when cell
count and wastewater concentration is low, compared
to high cell count and wastewater concentration. At
low cell count and wastewater concentration (nitrate =
90 mgL-1) C. vulgaris removed 69% of nitrate and
58% of COD within 10 days. When cell count and
wastewater concentration increased (nitrate = 178
mgL-1) C. vulgaris removed 80% of nitrate and 63% of
COD within 10 days suggesting that this alga is more
efficient in wastewater with high concentrations.
count at the day 8th which was significantly higher
than the other treatments. Overall, Scenedesmus
treatment, particularly S0 treatment, was more
efficient in biomass production, chlorophyll value and
nutrient uptake compared to Chlorella treatment.
Chlorella treatment was superior to the other
treatments in wastewater treatment, except for
phosphate uptake, which C0 treatment completely
depleted the medium phosphate content. Finally, it is
suggested that change in wastewater concentration
and algae type can improve municipal wastewater
refining.
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4