Journal of Water Process Engineering 36 (2020) 101274

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Journal of Water Process Engineering

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Municipal wastewater treatment by semi-continuous and membrane algal- T

bacterial photo-bioreactors
Elham Aminia, Azadeh Babaeib, Mohammad Reza Mehrniac, Jalal Shayeganb,*,
Mohammad-Saeed Safdaria
a
Department of Chemical Engineering, Brigham Young University, Provo, Utah 84602, USA
b
School of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
c
School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Conventional wastewater treatment systems demand a high cost mechanical aeration. Using algal-bacterial
Algal-bacterial culture systems helps to reach a cost-efficient treatment method by eliminating mechanical aeration, since algae produce
Membrane bioreactor the oxygen needed for treatment process. In this study, two groups of experiments were performed for domestic
Microalgae wastewater treatment processes. For the first group, semi-continuous microalgae-bacterial photo-bioreactors
Photo-bioreactor
were cultivated. Chlorella Vulgaris and activated sludge (AS) were used as microalgae and bacterial inoculums,
Wastewater treatment
respectively. The effect of different algae and AS ratios on the chemical oxygen demand (COD), N-NH4+ and P-
PO43− removal was studied. The removal efficiency of COD was above 93 % for the three tested algae and AS
inoculum ratios (5:1, 1:1, and 1:5). The reactor with algae: AS inoculum ratio of 5:1 achieved the highest final N-
NH4+ and P-PO43− removal efficiencies (88.0 ± 1.0 % and 84.0 ± 1.0 %, respectively). Furthermore, the
highest biomass concentration (1.96 g L-1 from initial amount of 0.3 g L-1) was observed in the reactor with
algae: AS ratio of 5:1. The ratio of 5:1 (algae: AS) was found as the optimum ratio which promoted the co-
operation between microalgae and AS for nutrient removal. For the second group of experiments, the optimum
ratio of 5:1 (algae: AS) was used in a membrane bioreactor, and the results showed that this reactor enhanced the
final removal efficacies from 88 to 98 % and from 84 to 89 % for N-NH4+ and P-PO43-, respectively.

1. Introduction These systems establish a cheaper alternative to mechanical aeration

Biological methods in wastewater treatment are preferred due to be
more cost efficient and environmentally friendly [1–4]. The use of
microalgae-bacterial systems for both domestic and industrial waste-
water treatments has received a great deal of attention in recent years
[5]. They are more commonly used as an alternative bio-system mostly
in tropical and subtropical regions [1,6,7].
Conventional wastewater treatment systems based on AS demand a
high cost mechanical aeration. Therefore, introducing a more cost-ef-
fective method seems to be essential. Using algal-bacterial systems
helps to reach a cost-efficient treatment method by eliminating me-
chanical aeration. Under illumination, algae assimilate the nutrients
(NH4+, NO3− and PO43-) available in the wastewater, and simulta-
neously produce oxygen by photosynthesis needed for heterotrophic
bacteria to mineralize organic materials. In return, they utilize the
carbon dioxide released from bacterial respiration to complete photo-
synthetic cycle, thereby reducing the emission of greenhouse gases.
[8–12]. Eisenberg et al. [13] estimated energy savings of more than 50
% for an algal-bacterial system compared to a conventional wastewater
treatment process.
Chlorella and Scenedesmus are unicellular green algae which are
widely used in wastewater treatment [14]. Microalgae can grow under
autotrophic (with inorganic carbon source and continuous light), het-
erotrophic (inorganic carbon source and without light), and mixo-
trophic (both organic and inorganic sources and without continuous
light) growth conditions [13,15]. Municipal wastewater is a proper
source of nitrogen, phosphorous and other elements (e.g., Cu, Mn, Ca,
Fe, K, S) which makes it a great medium for microalgae’s growth
[11,16]. In microalgae-based wastewater treatment systems, the func-
tions of microalgae include absorption of hazardous heavy metals, O2
supply and nutrient uptake [12,17–19,]. Furthermore, algae photo-
synthesis increases the pH and the amount of dissolved oxygen (DO),
which results in indirect disinfection. Without appropriate treatment,
extra amounts of nitrogen and phosphorus in discharged wastewater

⁎
Corresponding author.
E-mail address: shayegan@sharif.edu (J. Shayegan).

https://doi.org/10.1016/j.jwpe.2020.101274
Received 4 August 2019; Received in revised form 22 March 2020; Accepted 25 March 2020
2214-7144/ © 2020 Elsevier Ltd. All rights reserved.
E. Amini, et al.
cause downstream eutrophication and damage the ecosystem [19].
Although, there are chemical and physical treatment technologies, they
consume a large amount of energy during the removal process [20].
Chemical treatment methods are not only costly processes, but can also
lead to secondary contamination of sludge byproduct. However, com-
pared with chemical and physical processes, nutrients can be removed
in a cheaper and safer way by using algae-based systems [17,18,21,22].
Accumulation of nitrogen and phosphorus into algal and bacterial
biomass increases in the case of using algal-bacterial system. Only 26 %
N-NH4+ was assimilated into biomass during the use of bacterial system
in order to treat 1 g acetonitrile. However, using algal-bacterial culture
led to assimilation of 54 % N-NH4+ into biomass, under the same
condition [10,23]. The initial inoculum ratios of algae and bacteria may
affect the symbiosis of algae and bacteria and then result in different
nutrient removal efficiencies [24,25]. Guieysse et al. [12] showed that
the initial inoculation ratio of Chlorella Sorokiniana and Ralstonia Basi-
lensis has a significant impact on the salicylate degradation efficiency in
closed systems. AS has higher organic nutrient removal ability than the
normal bacteria included in municipal wastewater. Therefore, AS can
be used as bacterial inoculum in algal-bacterial based domestic waste-
water treatment systems to promote the nutrient removal efficiencies
[24].
Continuous microalgae cultivation in wastewater for nutrient re-
moval and biomass production for biofuel has been carried out in both
photobioreactors and open ponds [26–28]. One of the biggest concerns
of these systems is washout problems. Membrane bioreactors (MBR)
can address this problem by membrane filtration [26]. MBR have been
widely used recently because of their advantage of high sludge con-
centration, small footprint, and high effluent quality [25]. However,
high concentrations of mixed liquor suspended solids (MLSS) would
increase the non-Newtonian viscosities which can increase the aeration
energy consumption. Therefore, it is needed to develop an efficient
technique to reduce membrane fouling and decrease aeration cost in the
MBR system. There are some studies on combining algae system with
MBR to remove N and P in wastewater [29,30]. However, most of these
studies used external CO2 supply because of the absence of sludge
bacteria and resulted in slow nutrient removal efficiency of only algae
[25,31]. Furthermore, algae cells when using membrane filtration cause
dramatically fouling [32]. Previous researches have been shown that
combining algae-activated sludge and MBR decreases the aeration
consumption and the membrane fouling [25,33].
Many studies have achieved an excellent performance for different
algae/sludge inoculation ratios in batch mode. Ruiz-Martin et. al [34]
showed that, semi-continuous algal systems achieved higher removal
efficiencies than batch cultures. However, the nutrient removal per-
formance of semi-continuous algal-bacterial systems as well as the ef-
fect of algae/sludge inoculation ratio in these reactors is still unknown.
Therefore, the first objective of this work is to find the optimum ratio of
algae and AS in semi-continuous system to obtain the highest nutrient
removal efficiencies. Second, the optimum ratio is used in an algal-AS
MBR to enhance the removal efficiencies. During this research, the
experiments were conducted in 2 groups. The first group of experiments
was operated in semi-continuous algal-bacterial photo-bioreactors
(without mechanical aeration). Five different ratios of algae and AS
were used to find the optimum ratio that enhances the cooperation
between microalgae and AS for the removal of COD, ammonium and
phosphorous from municipal wastewater. For the second group of the
experiments, the optimum ratio of algae and activated sludge obtained
from the first group of experiments was used in a membrane bioreactor
to enhance the removal efficiencies.
2. Material and methods
2.1. Microorganisms
The algae inoculum (Chlorella vulgaris) was obtained through the
2
Journal of Water Process Engineering 36 (2020) 101274
Research Institute of Applied Science, Tehran, Iran. Microalgae were
pre-cultivated photo-autotrophically in sterile N-8 medium at ambient
temperature and were continuously illuminated by fluorescent lamps
under light intensity of 2000 lx. The aerobic AS was obtained from an
AS reactor in the Ekbatan municipal wastewater treatment plant in
Tehran, Iran. For adaptation, both microalgae and AS were fed with
synthetic municipal wastewater. Using synthetic medium not only
simplifies the use for initial laboratory–based experiments, but also
makes it easy to analyze major components without considering un-
known variables such as biotic variables [35].
2.2. Experimental operation
The first group of the experiments was carried out in semi-con-
tinuous culture in stirred cylindrical glass reactors with working vo-
lume of 650 mL for 21 days. For this study, semi-continuous experi-
ments were conducted with a fixed hydraulic retention time (HRT) of 7
days in all runs which was maintained by feeding 93 mL of feedstock
and removing 93 mL of effluent daily. An HRT between 7 and 10 days
and room temperature are found to be the optimum operating condition
for microalgae-bacterial systems [8]. Five different mass ratios of algae
and AS inoculum were considered. The features of the reactors are
shown in the Table 1. The reactors were operated in the same condition
and ambient temperature (around 25 °C) and were illuminated by
fluorescent lamps (with a light density of 2000 lx) under a photoperiod
of 12:12-h light/dark. The initial biomass concentration was chosen as
0.3 g L−1 according to the algal biomass concentration in common
microalgal wastewater treatment processes. Magnetic stirrers were used
as mixers with a mixing velocity of 300 rpm to avoid light limitation,
the sedimentation of biomass, and nutrient gradients. Therefore, the
photo-bioreactors can be considered as completely mixed reactors. The
velocity of 300 rpm was chosen due to its higher nutrient removal ef-
ficiency as well as higher algae growth rate [9].
The second part of the experiments was performed in a cylindrical
submerged membrane bioreactor with a working volume of 2.5 L using
the optimum ratio obtained from the first group of the experiments
(Supplementary Fig. S1) [27]. A circle membrane module with a hy-
drophilic Millipore mixed cellulose ester (MCE) with a pore size of 0.2
μm was used. The module was vertically submerged in the center of the
bioreactor. The reactors operated at constant permeate flux (17.3 LMH)
to provide a HRT of 3 days which was controlled with a peristaltic
pump. The reactor was fed continuously with the synthetic wastewater
and the feed flow rate was controlled by a level controller. The reactor
was illuminated with three cool-light fluorescent lamps with a total
intensity of 6000 lx which was fixed around and outside the cylinder.
The reactor was operated under room temperature of 25 °C.
Synthetic municipal wastewater was used as feed for all the reactors
(Supplementary Table S1). Glucose, NH4Cl, KNO3 and KH2PO4 were
used as organic carbon, ammonium, nitrate and phosphate sources,
respectively. The value and content of other required elements for
microorganisms’ growth were the same with N8 medium. pH was
controlled and maintained at a constant value of about 7.0 ± 0.3 by
adding base and acid [36].
Table 1
The features of the reactors.
Reactor Initial concentration of Initial concentration of Microalgae: AS
microalgae (g L−1) AS (g L−1) ratio
1 0.3 0 Only microalgae
2 0.25 0.05 5:1
3 0.15 0.15 1:1
4 0.05 0.25 1:5
5 0 0.3 Only sludge
E. Amini, et al.
2.3. Analytical procedures
pH was determined near the center of the reactors by pH meter
probe (240, ISTEK, Korea). DO was measured using a DO sensor
(WTW340i, Germany). For further analysis, such as COD, phosphate,
ammonium, and biomass concentration, the samples of 20 mL were
collected every 24 h. 20 mL feed was added to each reactor to com-
pensate for the removed sample. For determining the biomass con-
centration, the samples were filtered through 0.45-μm cellulose acetate
filters, dried in an oven at 105 °C for 2 h until the weight of the filters
remained stable, then weighted to calculate the dry weight of the
samples [15,37]. After filtration, the liquid samples were prepared for
analysis after eliminating some of the remained cells. The concentra-
tions of COD, N-NH4+ and P-PO43− (mg L-1) of liquid samples were
measured based on standard methods [38]. Dry weight and biomass
concentration of the reactor with only microalgae were measured
through the calibration curve using optical density of liquid samples
obtained from spectrometer (UNIC 2100) at 580 nm of wavelength
[15].
2.4. Determining the specific growth rates and doubling times
In the exponential phase of the microorganism’s growth profile, the
number of living cells doubles regularly with time. During this period,
the specific growth rates (μ) and doubling times (td) were calculated by
the Eqs. 1 and 2, respectively. td is the time required for cell population
to double [39].
ln n/ no
μ=
t − to (1)
ln2
td =
μ (2)
where to are the initial time and n and no are the cell concentrations at
times t and to·
2.5. Statistical analysis
All analytical tests (except DO measuring) were done in triplicate.
Comparison between the treatments was conducted in Sigma Plot 11.0.
Statistical analyses were performed for the biomass concentration, COD
and nutrient removal using one-way analysis of variance (ANOVA) (p
value < 0.05). In order to determine which system had a significant
difference with others, Holm-Sidak test was conducted for every binary
combination of the systems.
3. Results and discussion
3.1. COD removal
The COD removal efficiencies were calculated for all the reactors
and the results are presented in Fig. 1. The final removal efficiencies of
96 %, 95 %, and 97 % were observed in the mixed reactors with algae/
AS ratio of 5:1, 1:1 and 1:5, respectively. According to statistical ana-
lysis, the removal efficiencies in these reactors were not significantly
different (p value = 0.115), reflecting the fact that the initial algae and
AS inoculum ratios have no significant impact on COD removal effi-
ciency. Only microalgae and only sludge reactors were not able to
eliminate the COD more than 77 % and 80 %, respectively. The low
amount of removal in these reactors showed the importance of co-
operation between microalgae and sludge on COD elimination. Because
of the lack of sludge as the main CO2 producer in only algae reactor,
organic carbon decomposition did not take place successfully, and re-
sulted in low removal efficiency. The results of this study were com-
parable with literature. Su et al. [9] showed that COD removal effi-
ciencies were more than 90 % in the reactors with different ratios of
3
Journal of Water Process Engineering 36 (2020) 101274
algae and AS. In addition, the reactors with only algae and only sludge
reached the removal efficiencies less than 74 % [9]. Using Chlorella
Sorokiniana cultured heterotrophically, Kim et al. [36] could remove 40
and 80 % of COD from wastewater with ammonia concentration of 10
and 20 mg L−1, respectively. Comparing the removal efficiencies for
mixed algae-AS reactors investigated in this study with COD removal
efficiencies in batch cultures reported by Su et al. [9], indicated that the
batch and semi-continuous mixed photo-bioreactors could remove COD
over 90 % regardless of the initial inoculum ratio of algae and AS.
However, higher removal efficiencies were resulted from semi-con-
tinuous reactors used in this study.
3.2. Ammonium removal
N-NH4+ removal efficiencies were calculated, and the results are
presented in Fig. A. The reactor with algae/ AS ratio of 5:1 showed the
highest final removal efficiency of 88.0 ± 1.0 % due to preference of
microalgae to use ammonium as its main nitrogen source for auto-
trophic and heterotrophic growth. This reactor reached the highest
biomass production due to the proper cooperation between microalgae
and AS, and subsequently enough available CO2 for algae’s autotrophic
growth (Supplementary Fig. S2).
N-NH4+ Removal efficiencies of 70.0 ± 2.0, 29.5 ± 1.6, 79.3 ± 1.5,
and 64.3 ± 1.2 % were obtained for the reactors with only microalgae,
only sludge, 1:1, and 1:5 (algae/ AS), respectively. One-way ANOVA
indicated that N-NH4+ Removal efficiencies were significantly different
for different algae/AS ratio (p value < 0.05). Despite the lack of AS as
the main CO2 producer in the reactor with only microalgae, this reactor
could remove more than 75 % N-NH4+. The main reason is that the
organic carbon (COD) was used by microalgae as carbon source under
darkness condition for mineralizing ammonium and heterotrophic
growth [36]. The reactor with only sludge could not remove more than
30 % ammonium. The main reason is that nitrification is a slow process
[40] and the results of nitrogen removal indicate that this AS did not
contain a significant portion of effective nitrification bacteria. Also,
sludge does not have high ability in ammonium removal as microalgae
[24]. The mixed reactors with algae/ AS ratio of 1:1 and 1:5 could not
remove ammonium as high as the reactor with algae/ AS ratio of 5:1,
which indicated that initial inoculum ratio of microalgae and AS af-
fected the N-NH4+ removal. Therefore, it is concluded that the higher
the algae’s initial inoculum ratio, the higher the ammonium removal
efficiency. According to Pourasgharian Roudsari et al. [37], the addi-
tion of AS to microalgae in different mass ratios increased the N-NH4+
removal efficiencies in algae-AS based municipal wastewater treatment
systems in batch mode. In a previous study, it has been shown that the
reactor with algae/ AS ratio of 5:1 in batch mode had the highest and
fastest ammonium removal efficiency within 10 days [9]. However,
compared with batch culture [23], the higher removal efficiency of the
reactor with algae/ AS ratio of 5:1 indicated that semi-continuous
mixed algae/ AS photo-bioreactors have higher ammonium removal
abilities than batch cultures, which is consistent with the literature
where tested microalgae in semi-continuous mode was more effective in
removing N than batch cultures [39]. According to statistical analysis
(P value < 0.05), the significant nitrogen removal efficiency of the re-
actor 5:1 (algae/ AS) in both batch and semi-continuous cultures
showed that this ratio could be the best to promote cooperation be-
tween algae and AS for higher nutrient removal efficiency. The higher
removal efficiency of the reactor with algae/ AS ratio of 1:5 than the
reactor with only algae indicated that adding microalgae even in low
ratio to AS could increase the ammonium removal in wastewater
treatment systems without mechanical aeration. Compared to the ratios
of 5:1 and 1:1 (algae/ AS), the ratio of 1:5 of algae/ AS seemed to be
unsuitable to improve cooperation between algae and AS for nitrifica-
tion. The low initial inoculum ratio of microalgae in the reactor 1:5
could not provide enough oxygen for nitrification process.
E. Amini, et al.
Fig. 1. Final COD removal efficiencies (after stabilization) in the reactors with different ratios of algae and AS. The means followed by the same letter did not differ
significantly from each other (P value of 0.252 and 0.115 for a and b letter, respectively). Error bars represent the standard deviation of three replicate analysis tests.
3.3. Phosphorus removal
The daily phosphorous removal efficiencies are shown in Fig. 2B.
Statistical analysis of phosphorous removal efficiencies, using one-way
ANOVA (Holm-Sidak test), indicated a significant difference between
different ratios of algae/AS (p value < 0.05). The highest final phos-
phorous removal efficiency was observed in the reactor with algae/AS
ratio of 5:1 (84.0 ± 1.0 %), followed by the reactor with algae/AS ratio
of 1:1 (72.2 ± 1.1 %). The other mixed reactor with algae/AS ratio of
1:5 reached a final removal efficiency of 60.1 ± 1.3 %.
The reactor with only microalgae could not remove more than
51.2 ± 1.4 % phosphorous despite the high capability of microalgae in
mineralizing phosphorous. Two possible reasons may explain this: (1)
light inhibition due to high concentration of microalgae cells prevented
the optimum autotrophic growth of microalgae [24], (2) because of the
lack of AS as the main CO2 supply in the reactor, there was not enough
CO2 for microalgae photosynthesis. Despite the lack of AS, the micro-
algae were able to remove this amount of phosphorous. This could be
explained by considering the ability of chlorella to switch its metabolism
between autotrophic and heterotrophic depending on carbon source
and darkness or lightness condition [15]. Heterotrophic microalgae
used COD as carbon source under darkness condition to mineralize
phosphorous. In addition, by using COD, heterotrophic microalgae
produce CO2 under darkness and this CO2 could be used by autotrophic
microalgae under light.
The reactor with only sludge had the lowest final removal efficiency
of 30 % due to the lack of microalgae and very poor phosphorous re-
moval capability of AS [24]. In addition, the reactor with only sludge
had the lowest pH level. Therefore, abiotic phosphorous precipitation
did not take place [11,41].
Mixed algae/ AS reactors showed better performance in removing
phosphorous than only algae and only sludge bioreactors. By increasing
the amount of microalgae, higher removal efficiency was observed. The
highest removal efficiency achieved by the reactor with algae/ AS ratio
of 5:1 indicated that in this ratio, microalgae and AS have the best
cooperation for P-PO43− removal.
Compared with batch cultures [23], semi-continuous mixed algae/
AS photo-bioreactors had higher phosphorous removal abilities than
batch cultures, which is consistent with the literature where tested
microalgae in semi-continuous mode was more effective in removing P-
PO43− than batch cultures [39].
4
Journal of Water Process Engineering 36 (2020) 101274
3.4. Biomass concentration in the reactors
The growth rates and doubling times for exponential growth phase
were calculated by Eq. 1 and Eq. 2, respectively (Table 2). One-way
ANOVA (Holm-Sidak test) indicated that the mixed algae/AS reactors
had significantly higher biomass concentration than only algae and
only sludge reactors (p value < 0.05) due to cooperation between algae
and AS (Supplementary Fig. S2). Microorganisms assimilate the nu-
trient for their growth. Then, there is a direct relation between biomass
production and nutrient removal in wastewater treatment reactors.
The mixed reactors achieved higher nutrient removal efficiencies
than only algae and only sludge reactors. The optimum reactor (5:1)
which achieved the highest nutrient removal efficiency had the highest
biomass productivity rate of 0.143d−1, lowest doubling time of 4.82 d,
and the highest biomass concentration. The 1:1 and 1:5 (algae/AS)
reactors, respectively, had the next highest removal efficiencies and
growth rates. These results showed that a higher growth rate leads to
higher nutrient removal efficiency. In addition, in algae/AS system, the
initial concentration of activated sludge has an influence on the growth
of C. vulgaris [42]. A higher initial inoculum ratio of microalgae in
mixed algae/AS reactors sped up the microorganisms’ growth. Very
high concentration of activated sludge was not suitable for algal growth
[42]. The reactors with algae/ AS ratio of 5:1 and 1:1 reached slightly
higher growth rates than the 1:5 culture.
3.5. DO variation under different ratios of microalgae and activated sludge
The variations of DO in the reactors with different algae/ AS in-
oculum ratios are shown in Fig. 3. For the reactor with only sludge, DO
was always at low level because of the lack of microalgae as the main
oxygen producer. In the reactor with only microalgae, DO had an in-
creasing trend for 21 days. A possible reason for this high DO con-
centration is the lack of AS as the main oxygen consumer in the reactor,
so the produced oxygen by microalgae through the photosynthesis
process was accumulated and reached 13 mg L−1 on the last day of the
experiments. According to Su et al. [24], high oxygen level was ob-
served in the reactor with only algae in batch culture. The results of
both batch and semi-continuous cultures showed that the reactor with
only microalgae can produce high amount of DO without mechanical
aeration. Chlorella can switch its metabolism between photoautotrophic
and heterotrophic [43]. Using the photoperiod of 12:12-hr light/dark
E. Amini, et al.
Fig. 2. (A) N-NH4+ and (B) P-PO43− removal efficiency in the reactors with different ratios of algae and activated sludge. Error bars represent the standard deviation
of three replicate analysis tests.
enabled microalgae to use photoautotrophic metabolism under illumi-
nation for photosynthetic process [15]. Under darkness period, micro-
algae changed its metabolism into heterotrophic and by using organic
carbon as carbon source could produce CO2 thorough photosynthesis
[15]. The produced CO2 (dissolved in water) was used by autotrophic
microalgae for photosynthesis. This cycle helped microalgae to produce
high amount of oxygen with no need for mechanical aeration. In the
reactor with microalgae/ AS ratio of 1:5, discernable decrease of DO in
5
Journal of Water Process Engineering 36 (2020) 101274
the beginning of the process was observed, which indicated that the
dominant population of AS at the beginning of the experiment caused
higher consumption of oxygen than what was produced. The other
possible reason for that is the high COD removal during the first days of
the experiment (Fig. 3). AS has a considerable ability to mineralize
organic carbon. Mineralizing organic carbon, AS consumed more
oxygen and caused DO concentration to decrease. After three days, the
biomass concentration in the reactor started to increase (Supplementary
E. Amini, et al. Journal of Water Process Engineering 36 (2020) 101274

Table 2 [9]. The longest and lowest duration times (7 and 12 days) of low DO
The growth rates and doubling times of the reactors (Average data during were observed in the reactors with algae/ AS ratio of 5:1 and 1:5 in
steady state condition). batch mode, respectively [9]. Compared with batch culture, semi-con-
Reactor (algae:AS) μ (d−1) td (d) Final concentration of biomass (g L−1) tinuous mixed algae/ AS photo-bioreactors never experienced the
duration time of low DO, because all the reactors were fed with fresh
Only algae 0.10 6.86 1.28 ± 0.01 synthetic wastewater every 24 h. This feature of the semi-continuous
5:1 0.14 4.82 1.94 ± 0.04
photo-bioreactors showed that biological wastewater treatment process
1:1 0.11 6.13 1.70 ± 0.01
1:5 0.10 6.79 1.59 ± 0.03 using algae-AS does not need the costly mechanical aeration.
Only sludge 0.09 7.07 1.41 ± 0.01

3.6. Removal efficiencies in the membrane bioreactor

Error bars represent the standard deviation of three replicate analysis tests.
Statistical analysis of biomass concentration, using one-way ANOVA (Holm-
Sidak test), indicated a significant difference between different ratios of algae/
AS (p value < 0.05).
Fig. S2). Increasing the concentration of DO showed that the amount of
microalgae increased, which indicated that symbiosis of microalgae and
AS promoted the microalgae’s growth.
This increasing amount of DO was continued until day 9. After that,
the DO concentration reached an almost constant level which indicated
that the amount of consumption and production of DO reached an
approximately fixed rate. At 1:1 reactor, in the beginning of the process
DO concentration was almost constant and after 13 days, the oxygen
rate was ascending which indicated that symbiosis of microalgae-sludge
facilitated the microalgae's growth and probably the microalgae po-
pulation was dominant. The difference between reactor 5:1 (algae/ AS)
and 1:1 is that because of the dominant population of microalgae in the
reactor with algae/AS ratio of 5:1, the oxygen level was ascending at
the beginning. The amount of DO in the reactor with algae/ AS ratio of
5:1 was more than the reactor with only algae in the first days of the
experiment. One possible reason for this high level of DO is the better
condition of the microalgae in the 5:1 reactor due to the presence of AS
as the main CO2 producer. In a previous study by Su et al. [9], reactors
with different ratios of algae and AS had distinct level of oxygen during
the experiment in batch culture. All of the reactors experienced a
duration time of low DO and after that the oxygen level started to go up
The results obtained from the first group of the experiments showed
that the ratio of 5:1 (algae/AS) is an optimum ratio for proper co-
operation between algae and AS for P-PO43− and N-NH4+ removal.
This ratio was used in the membrane bioreactor, and the removal ef-
ficiencies were compared with the results obtained from semi-con-
tinuous reactor and a similar study by Sun et al. [25]. Fig. 4 shows the
final P-PO43− and N-NH4+ removal efficiencies obtained from this
study using membrane and semi-continuous reactors and from litera-
ture using membrane bioreactor with algae/ AS ratio of 5:1. It can be
seen that, using membrane bio-reactor enhanced the removal effi-
ciencies from 88 to 98 % and from 84 to 89 % for N-NH4+ and P-PO43−,
respectively. This phenomenon is attributed to the high shear force
(aeration for preventing membrane fouling) in MBR. Previous studies
have shown that the amount of extracellular polymer substances (EPSs)
production was promoted by higher shear force. Furthermore, EPS can
stimulate the uptake of N by microalgae [25,26]. Therefore, in this
study, the nitrogen removal efficiency increased in MBR. Comparing
the results from this study with the removal efficiencies obtained by
Sun et al. [25] using membrane reactor with a ratio of 1:1, showed that
the membrane reactor containing 5:1 ratio of algae/AS used in this
study had a better performance and reached higher removal efficiencies
due to high ability of microalgae in nitrogen removal. However, in
previous study by Sun et al. [25], the HRT was 12 h which was different
than current study (3 days). To have a better understanding about the
effect of HRT and algae/AS ratio on removal efficiencies, a future work

Fig. 3. Dissolved oxygen concentration in the reactors with different ratios of algae/ AS.

6
E. Amini, et al.
Fig. 4. Final N-NH4+ and P-PO43− removal efficiencies obtained from this study using semi-continuous and membrane bio-reactors with algae/ AS ratio of 5:1 and
literature using membrane bio-reactor with algae/ AS ratio of 1:1.
should be conducted with an algae/AS ratio of 1:1 at an HRT of 12 h.
It can be concluded that using MBR containing algae/AS is suitable
for the nutrient removal from the wastewater. The main problem is
membrane fouling that should be further studied to control the fouling
in wastewater treatment systems.
Although, novel results were obtained from this research, future
works need to be done studying the effect of operating condition on
nutrient removal performance. Temperature and HRT affect the growth
rate and nutrient removal performance. Low to negative growth rates
were observed in microalgal cultures operating at lower temperatures
[7,8]. Using the optimum ratio obtained from this study, the effect of
temperature, photo period and different HRT should be examined in
future to find an optimum operating condition.
4. Conclusion
Semi-continuous algal-bacterial photo-bioreactors were successfully
used to treat domestic wastewater. Five different ratios of algae and AS
(5:1, 1:1, 1:5, only algae and only sludge) were chosen. Chlorella
Vulgaris and aerobic AS were used as microalgae and bacterial in-
oculums. The results indicated that the different initial inoculum ratios
of algae/ AS had significant effects on ammonium and phosphorus re-
moval. The COD removal efficiencies were more than 90 % regardless
of microalgae and AS inoculation ratios. Eliminating the need for me-
chanical aeration, algae/ AS wastewater treatment cultures reached the
high nutrient removal. The ratio of 5:1 (algae/AS) was found to be the
best ratio for proper cooperation between algae and AS for P-PO43− and
N-NH4+ removal. Furthermore, this study showed that, semi-con-
tinuous mixed algae/ AS photo-bioreactors had higher nutrient removal
efficiencies than batch cultures. Finally, the optimum ratio of 5:1
(algae/ AS) were used in a membrane bioreactor and the results showed
that this reactor enhanced the removal efficacies.
Declaration of Competing Interest
The authors declare that they have no competing interests
Funding
This work was supported by Iran National Science Foundation
(INSF) (Grant number 97001687).
7
Journal of Water Process Engineering 36 (2020) 101274
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jwpe.2020.101274.
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