Bioresource Technology 323 (2021) 124629

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Comparing the use of a two-stage MBBR system with a methanogenic MBBR

coupled with a microalgae reactor for medium-strength dairy
wastewater treatment
Eirini Zkeri , Athanasia Iliopoulou , Alexandra Katsara , Angeliki Korda , Maria Aloupi ,
Georgia Gatidou , Michail S. Fountoulakis , Athanasios S. Stasinakis *
Water and Air Quality Laboratory, Department of Environment, University of the Aegean, 81100, Greece

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Two different systems were compared

for medium strength dairy wastewater
treatment.
• >93% removal of COD, NH4-N, TKN,
partial removal of PO4-P in AnMBBR-
AeMBBR system.
• Full COD removal, partial removal of
other pollutants in AnMBBR-microalgal
SBR.
• Energy autonomy of the AnMBBR at
ambient conditions, use as first-
treatment step.
• High protein content of microalgae after
acclimatization to dairy wastewater.

A R T I C L E I N F O A B S T R A C T

Keywords: Two systems were compared for medium-strength dairy wastewater treatment. The first comprised a meth­
Milk processing wastewater treatment anogenic Moving Bed Biofilm Reactor (AnMBBR) and an aerobic MBBR (AeMBBR), while the second an AnMBBR
Biocarriers and a sequencing batch reactor (SBR) with Chlorella sorokiniana. The AnMBBR, under ambient conditions,
Microalgae
achieves biogas production sufficient enough to attain energy autonomy. The produced energy was 0.538 kWh
Biogas production
m− 3, whereas the energy consumption 0.025 kWh m− 3. Its coupling with the AeMBBR removed COD, NH4-N
Biomass characteristics
TKN, and PO4-P by 93 ± 4%, 97 ± 3%, 99 ± 1% and 49 ± 15%, respectively, while the use of the SBR as a second
step eliminated totally COD but partially the other pollutants. The higher nitrogen removal in the first system
was due to nitrification occurring in the AeMBBR. The acclimatization of microalgae to dairy wastewater
enhanced their growth. Their protein content was 54.56%, while starch and lipids were 3.39% and 23.1%,
respectively.

1. Introduction wastewater in Europe and USA. It includes factories that transform the
raw milk to pasteurized or condensed milk and/or produce materials
The dairy industry is one of the main sources of agro-industrial such as cheese, butter, yoghurt, ice-creams, creams and milk or whey

* Corresponding author.
E-mail address: astas@env.aegean.gr (A.S. Stasinakis).

https://doi.org/10.1016/j.biortech.2020.124629
Received 26 November 2020; Received in revised form 22 December 2020; Accepted 25 December 2020
Available online 30 December 2020
0960-8524/© 2020 Elsevier Ltd. All rights reserved.
E. Zkeri et al.
powders (Slavon, 2017). The produced wastewater mainly consists of
milk and milk products residues, byproducts of the processing opera­
tions, water used for the cleaning of the equipment and sanitary
wastewater from the staff (Ahmad et al., 2019). Its quantitative and
qualitative characteristics may vary depending on the processing
methods used, the final products and the factory’s size. In general, the
two main types of wastewater produced in dairy industry are a) the high-
strength wastewater (e.g. cheese whey, milk permeate) that is charac­
terized by the very high concentrations of organic matter, lactose and
fats, reaching COD concentrations up to 100 g L− 1 and b) the medium-
strength dairy effluent (e.g. effluents from the production of yoghurt,
butter ice-creams, cheese final wastewater effluents) that does not
exceed 5 g L− 1 COD (Carvalho et al., 2013; Slavov, 2017) and will be
studied in the current article.
After primary treatment that aims to the removal of fats and sus­
pended solids, activated sludge process is mainly applied for the sec­
ondary treatment of low- and medium-strength dairy effluents (Buntner
et al., 2013; Goli et al., 2019). Despite its wide use, problems such as the
poor sludge settling and the inefficient removal of organic compound
are often observed (Ahmad et al., 2019). Additionally, this process is
characterized by high operational costs and energy consumption, sig­
nificant sludge production and increased greenhouse gas emissions
(Mamais et al., 2015; Goli et al., 2019). To overcome such problems,
new treatment technologies should be developed and tested in order to
achieve sustainable dairy wastewater management that will combine
pollution prevention and resource recovery under the concept of circular
economy (Ahmad et al., 2019; Wang and Serventi, 2019).
Among them, aerobic and methanogenic Moving Bed Biofilm Re­
actors have been widely used for the treatment of municipal and in­
dustrial wastewater (di Biase et al., 2018; Vyrides et al., 2018). In these
systems, the use of floating biocarriers allows the growth of high con­
centrations of attached bacterial biomass per m3 of reactor achieving
efficient removal of organic carbon, high nitrification rates and impor­
tant micropollutants’ removal (Mazioti et al., 2015; Torresi et al., 2016).
Additionally, the combination of strictly anaerobic and aerobic condi­
tions in series allows the simultaneous energy recovery through the
biogas production (Gu et al., 2017; Kora et al., 2020). The aforemen­
tioned characteristics create the ideal conditions for their use in the
treatment of medium strength industrial wastewater. Beside the above,
limited data is so far available on the use of MBBR systems in dairy
wastewater treatment, while no information has been published about
the use of methanogenic MBBR and the production of biogas. In an early
study, Andreottola et al. (2002) used an aerobic MBBR system and re­
ported 80% removal of COD at an organic loading of 5 kg COD m− 3 d− 1,
while nitrogen removal ranged between 13.3 and 96.2%. Luostarinen
et al. (2006) used an aerobic MBBR for the post-treatment of a mixture of
dairy wastewater, kitchen waste and black water that had been anaer­
obically treated and concluded that the combination of the anaerobic
sludge blanket (UASB) reactor and the MBBR was able to remove 92% of
COD and >65% of nitrogen.
Since 2010, a large scientific effort has also been directed towards
the development of microalgal systems for wastewater treatment. The
use of microalgae under mixotrophic conditions can achieve simulta­
neously organic carbon, nitrogen and phosphorus removal from waste­
water (Mohsenpour et al., 2021), while the valorization of produced
biomass can provide an extra revenue (Nagarajan et al. 2020). On the
other side, comparing to the growth of microalgae in freshwater media,
there are several points that should be overcome in cases of agro in­
dustrial wastewater treatment, such as the possible surplus of organic
loading, the presence of organic compounds that are not assimilated by
the microalgae and the required pretreatment for assuring system’s
stability (Gupta et al., 2019; Mohsenpour et al., 2021). Concerning dairy
wastewater, so far several microalgal species have been tested under
different experimental conditions for their treatment (Pandey et al.,
2019; Zhu et al., 2019). Among them, Chlorella sorokiniana is a small,
fast growing, robust microalgae that can be grown under mixotrophic
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Bioresource Technology 323 (2021) 124629
conditions (Lizzul et al., 2018; Kotoula et al., 2020). Hena et al. (2015)
cultivated Chlorella sorokiniana in dairy farm effluents and observed
important growth and lipids accumulation under photoheterotrophic
conditions. Recently, Asadi et al. (2019) conducted batch experiments
using preliminary and secondary treated dairy wastewater and reported
significant removal of COD and nutrients in a period of 4 to 8 days.
Based on the above, the main objective of the current work was to
compare at lab scale the use of two different novel systems that apply
MBBR or/and microalgae technology for the treatment of medium-
strength dairy wastewater. The first system comprised a methanogenic
MMBR connected in series with an aerobic MBBR, while the second
included a methanogenic MBBR and a sequencing batch reactor (SBR)
with Chlorella sorokiniana that operated with no CO2 enrichment and pH
adjustment. An energy analysis was conducted for the methanogenic
MBBR to investigate its autonomy and its potential to act as a pre­
treatment step for dairy wastewater treatment under ambient conditions
while the protein, lipid and carbohydrate content of the produced
microalgal biomass was determined to identify possible valorization
options. The strong points and the limitations of the different technol­
ogies were discussed. To the best of our knowledge, this is the first study
that examines these combinations of technologies for dairy wastewater
treatment.
2. Materials and methods
2.1. Supply of dairy wastewater
Dairy effluents were collected every two weeks for a period of five
months in 2019 from the outlet of the dissolved air flotation tank of a
dairy factory producing feta cheese, cottage cheese and yoghurt in
Lesvos Island (Greece). The wastewater consisted of a mixture of final
whey effluents, water used for the cleaning of the equipment and sani­
tary wastewater. After collection, they immediately transferred to the
lab, sieved through a 1 mm mesh and stored at − 20 ◦ C until use. Their
characteristics are shown in Table 1 (2nd column).
2.2. Experiments in the methanogenic – Aerobic MBBR continuous-flow
system
A lab-scale system consisting of a strictly anaerobic (methanogenic)
moving bed biofilm reactor (AnMBBR) and an aerobic moving bed
biofilm reactor (AeMBBR) was used for dairy wastewater treatment.
This system had recently been used for the treatment of municipal
wastewater and a mature biofilm had developed on the biocarriers (Kora
et al., 2020). The 3-L AnMBBR was airtight sealed and it was filled by
40% (v/v) with sponge cubic form carriers (Nisshinbo Chemical Inc.).
The effluent of the AnMBBR was transferred via natural flow to a flask
chamber connected with a bag for biogas collection and with AeMBBR
for further wastewater treatment. The aerobic reactor had a volume of
3.8 L and it was filled by 33% (v/v) with K3 type biocarriers (Anox­
Kaldnes). Aerobic conditions (DO > 4 mg L− 1) along with efficient
mixing of the biocarriers were assured using air-stone diffusers in the
AeMBBR, while mechanical stirring was used in the AnMBBR.
Table 1
Characteristics of the influent and effluent dairy wastewater from the meth­
anogenic – aerobic MBBR system used in the current study (values have been
expressed as mean ± sd). The applied organic loading rate (OLR) was equal to
2.45 ± 0.87 kg COD m− 3 d− 1.
Influents Effluents of Effluents of
AnMBBR AeMBBR
pH 7.75 ± 0.60 7.34 ± 0.26 8.24 ± 0.11
CODdis (mg L− 1) 2499 ± 812 1657 ± 528 167 ± 99
NH4-N (mg L− 1) 89.1 ± 34.8 91.3 ± 27.7 2.7 ± 1.4
TKN (mg L− 1) 120.0 ± 10.0 137.6 ± 19.9 3.7 ± 0.4
PO4-P (mg L− 1) 20.7 ± 9.6 20.5 ± 10.4 11.0 ± 4.5
E. Zkeri et al.
The system was operated for a period of three (3) months and the
applied organic loading ratio (OLR) was equal to 2.45 ± 0.87 kg COD
m− 3 d− 1. The hydraulic retention time (HRT) was equal to 24 h for the
AnMBBR and 28.8 h for the AeMBBR. In order to investigate biogas
production and system’s performance when the AnMBBR is not heated
but operates under ambient conditions, no temperature adjustment was
conducted during the experiment. As a result, the temperature in the
MBBR system was equal to 18 ± 2 ◦ C. At the start of the experiment, the
system was fed with dairy wastewater for a period of two weeks in order
to assure acclimatization of the microorganisms to the substrate. Af­
terwards, samples were periodically taken from the influents and ef­
fluents of each reactor and analyzed for COD, NH4-N, Total Kjeldahl
Nitrogen (TKN) and PO4-P to monitor its performance. The temperature
and pH were monitored on a daily basis, while biogas volume was
continuously collected and measured once a week (as a weekly average).
2.3. Acclimatization of Chlorella sorokiniana to dairy wastewater and
batch experiments
Chlorella sorokiniana UTEX 1230 was taken by Culture Collection of
Algae and Protozoa (CCAP, Scotland, UK) and cultivated in growth
medium (3 N-BBM + V) for 21 days (OECD, 2011). Stock solutions were
initially prepared in biologically treated municipal wastewater as
described in our previous paper (Kotoula et al., 2020), while afterwards,
Chlorella was gradually acclimatized to 40% filtered dairy wastewater
for a period of 27 days. 200-mL cotton-gauze plugged flasks containing
2 mL of exponentially growing cells of Chlorella sorokiniana and 98 mL of
the appropriate dilution of dairy wastewater with secondary treated
wastewater were used for the acclimatization of the biomass. The
growth of the microalgae was monitored using optical density (OD) as a
proxy for cell number and a conversion factor of 211.6 was applied for
converting OD measurements to biomass concentration (mg L− 1) as re­
ported by Gatidou et al. (2019).
Two sets of batch experiments were conducted afterwards with
Chlorella sorokiniana to study the effect of light duration (Experiment A:
24 h per day light versus 16 h light – 8 h dark), and biomass acclima­
tization (Experiment B: acclimatized versus non-acclimatized microalgal
biomass to dairy wastewater) on the growth of microalgae and their
efficiency to remove the main pollutants from dairy wastewater. The
experiments lasted 7 d, they were conducted in triplicate with no pH
adjustment in cotton-gauze plugged flasks containing 250 mL of expo­
nentially growing cells of Chlorella sorokiniana and 350 mL of anaero­
bically treated dairy wastewater taken from the outlet of the AnMBBR.
Samples were taken at Days 3, 5 and 7 and they were analyzed for COD,
NH4-N, TKN, PO4-P and OD.
Both acclimatization of biomass and batch experiments were per­
formed in a temperature-controlled chamber (24 ± 2 ◦ C, continuous
illumination with OSRAM, FQ 39 W/840 HO fluorescent lamps). To
prevent cells’ clumping, the flasks with microalgae were manually
shaken on a daily basis.
2.4. Experiments in the methanogenic MBBR – Microalgae SBR system
In these experiments, the performance of a system consisted of the
AnMBBR and a sequencing batch reactor containing microalgae was
evaluated. The AnMBBR operated at operational conditions similar to
those described in Paragraph 2.2. The effluents of this reactor were
collected and transferred during the study to a 2-L flask containing
Chlorella sorokiniana that operated under sequencing batch conditions.
At the start of the experiments, the SBR was inoculated with 800 mL of
exponentially growing cells of Chlorella sorokiniana that had been
acclimatized to anaerobically treated dairy wastewater, while 1.2 L of
filtered dairy effluents of the AnMBBR were added. Mixotrophic con­
ditions (16 h light/8h dark) were applied using four fluorescent lamps
that had been arranged around the SBR (Kotoula et al., 2020), while
seven (7) experimental cycles were run (duration of each cycle: 3 d).
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Bioresource Technology 323 (2021) 124629
Upon completion of each experimental cycle, 1500 mL of treated
wastewater was removed from the SBR, while an equal volume of
wastewater was added. Samples were taken at the start and end of each
experimental cycle from the SBR and they were analyzed for COD, NH4-
N, TKN, PO4-P and OD. At the end of the experiment, the produced
biomass was also collected and analyzed for proteins, starch and lipids.
The temperature in the SBR during the experiments was equal to 26 ±
2 ◦ C.
2.5. Analytical methods
COD, NH4-N, TKN and PO4-P were measured according to Standard
Methods (APHA, 2005). Specifically, the closed reflux colorimetric
method was used for COD; NH4-N was measured with titrimetric method
(after distillation); TKN after digestion, distillation and titration of
ammonium nitrogen and PO4-P with the ascorbic acid method. Tem­
perature and pH were measured using portable instruments. Before
spectrophotometric analyses, samples were centrifuged and filtered
(Millipore membrane filters, 47 mm diameter, 0.45 µm pore diameter)
for removing color and solids. OD was measured at 550 nm using a 6405
UV/Vis spectrophotometer (Jenway, UK), while the volume of the pro­
duced biogas from the AnMBBR was determined by water displacement.
Concerning the characterization of microalgal biomass, proteins were
determined according to AOAC Official Method 2001.11 (AOAC Inter­
national, 2002; Thiex et al., 2002), lipids according to the gravimetric
method of D’Oca et al. (2011), while the anthrone method was used for
the determination of starch (Hansen and Møller, 1975), as modified by
Marshall (1986).
2.6. Energy balance analysis, other equations and statistical treatment
An energy balance analysis was performed for the lab-scale meth­
anogenic MBBR based on methodology described by similar previous
studies regarding methanogenic MBR systems (Mei et al., 2016; Xiao
et al., 2018; Chen et al., 2019). Specifically, the energy balance was
calculated as follows:
ΔE = Eo − (Em + Ep + Eh ) (1)
where ΔE is the energy balance, Eo is the energy production, Em is the
energy demand for mixing, Ep is the energy demand for pumping and Eh
is the energy demand for heating. All energy units are expressed as kWh
per m3 of wastewater.
The energy production (Eo) of the methanogenic MBBR was calcu­
lated from the biogas yield, according to Eq. (2):
Pbiogas × V × ξ × ηm
Eo = (2)
Q
where, Pbiogas is biogas yield (m3 biogas m− 3 reactor d− 1), V is the
working volume of reactor (m3), ξ is the lower heating value of biogas
(5.97 kWh m− 3), Q is the influent flow rate (m3 d− 1) and ηm is energy
conversion efficiency of biogas. It was assumed that 35% and 50% of the
energy in biogas could be converted into electricity and heat, respec­
tively (Lantz, 2012),
The energy consumed by an impeller for mixing was determined
through the following equation:
Np × ρ ( n )3
Em = × × D5 (3)
1000 × q × ζ 60
where, Np is the dimensionless power number, related to the Reynolds
number and shape of the reactor. The power number of an impeller for a
typical anaerobic cylindrical tank was assumed to be 0.75 (Meister et al.,
2018), ρ is the density of influent (1000 kg m− 3), q is the feed rate (m3
h− 1), ζ is the efficiency factor (0.75) of electrical energy conversion into
mixing energy (Ruggeri et al. 2010), n is the rotating speed of impeller
(60 r min− 1 in this experiment) and D is the diameter of impeller. It is
E. Zkeri et al.
mentioned that the stirrer of the examined reactor was in use for 15 min
every hour.
The energy production for pumping was estimated as follows:
Q×γ×h
Ep = (4)
1000 × n × q
where, Q is the influent flow rate (m3 d− 1), γ is 9800 N/m3, h is the
hydraulic pressure head (m), n is the pump efficiency (60%) and q is the
feed rate (m3 h− 1).
The energy required for raising the influent temperature to the
reactor temperature was calculated as Eq. (5)
ρ × Q × κ × (TAnMBBR − Ti )
Eh = (5)
Q
where, ρ is the density of influent (1000 kg m− 3), Q is the influent flow
rate (m3 d− 1), κ is specific heat of the influent (0.00116 KWh kg− 1 ◦ C− 1),
TAnMBBR is the temperature of the reactor and Ti is the temperature of the
influent. It was assumed that 80% of required heat could be recovered
(due to cyclic utilization) without considering the heat of biogas (Xiao
et al., 2018; Chen et al., 2019).
The removal of major pollutants in MBBR system as well as in the
experiments with microalgae was calculated according to Eq. (6):
Cin − Cout
%Removal = × 100 (6)
Cin
where, Cin and Cout are the concentrations at the influents and at the
effluents of the system or at the start and at the end of each experiment.
The specific growth rate of Chlorella sorokiniana in acclimatization
and batch experiments was calculated using Equation (7) (OECD, 2011):
μ=
lnXj − lnXi
(day− 1 ) (7)
tj − ti
where, μ is the specific growth rate, Xi is the biomass concentration at 0 h
and Xj is the biomass concentration at 72 h.
Statistical analysis was performed using Origin. The Kolmogorov-
Smirnov test was used to examine the normality of data and one-way
ANOVA was used for comparing major pollutants removal during
different experiments. Tukey’s post-hoc test was used for the determi­
nation of the significant differences between groups. All tests were run at
the 0.05 significance level and all comparisons mentioned hereafter are
based on the results of the statistical analysis.
3. Results and discussion
3.1. Operation of the methanogenic-aerobic MBBR system for the
treatment of medium-strength dairy wastewater
The two-stage MBBR system operated at an OLR value of 2.45 ± 0.87
kg COD m− 3 d− 1. The average dissolved COD concentrations of the in­
fluents where equal to 2499 mg L− 1, while the relevant values for NH4-
N, TKN and PO4-P were 89.1, 120 and 20.7 mg L− 1, respectively
(Table 1).
The pH of the influents was decreased in the AnMBBR possibly due to
the production of volatile fatty acids (VFAs) under anaerobic conditions.
On the other hand, an increase of pH to values higher than 8.2 (on
average) was observed at the final effluents of the AeMBBR (Table 1) due
to VFAs biodegradation under aerobic conditions. The total removal of
CODdis, NH4-N and TKN exceeded 93% during the study (Fig. 1), indi­
cating the ability of the system to remove these pollutants sufficiently
from dairy wastewater. As a result, the average concentrations of
CODdis, NH4-N and TKN lowered to 167, 2.7 and 3.7 mg L− 1 at the ef­
fluents of the system (Table 1). On the other hand, a partial removal of
PO4-P (49 ± 15%) was observed resulting to average phosphorus con­
centrations higher than 10 mg L− 1 at the outlet of the system (Fig. 1,
4
Bioresource Technology 323 (2021) 124629
Fig. 1. Removal of COD, NH4-N, TKN and PO4-P in the AnMBBR-AeMBBR
system used in the current study for the treatment of medium strength
dairy wastewater.
Table 1).
Concerning the role of different bioreactors on pollutants’ removal,
both reactors contributed significantly to the removal of COD, while the
removal of other major pollutants was solely observed in the AeMBBR
(Fig. 1). The contribution of both anaerobic and aerobic reactors on the
removal of organic loading has also been reported in previous studies
where anaerobic treatment was used as a first step for medium-strength
dairy wastewater treatment. Specifically, Luostarinen and Rintala
(2005) reported that the CODdis removal ranged between 33 and 62%
when treated in an one-phase UASB reactor operating at OLR values
ranging between 0.103 and 0.240 kg COD m − 3 d− 1. Tawfik et al. (2008)
used an UASB reactor followed by activated sludge for the treatment of
combined dairy and domestic wastewater and reported on average 69%
removal of total COD for OLR ranging between 1.9 and 4.4 kg COD m − 3
d− 1. Finally, Buntner et al. (2013) observed a total COD removal ranging
between 65 and 85% at the first anaerobic step of an UASB-MBR system
receiving synthetic dairy wastewater at a mean OLR of 1.95 kg m− 3 d− 1
and operating at ambient temperatures. Previous articles with meth­
anogenic MBBRs have also shown that these reactors cannot sufficiently
remove nitrogen and phosphorus and an aerobic step should be added if
there is a need to eliminate them (Gu et al., 2017; Kora et al., 2020).
Collection and measurement of the biogas volume from the AnMBBR
showed that an amount of 106 ± 126 mL per L of reactor and day was
produced during the study. The energy balance calculations showed that
the operation of the AnMBBR under ambient temperature (18 ◦ C)
resulted to total energy production of 0.538 kWh m− 3, while the total
energy consumption was quite low (0.025 kWh m− 3) (Table 2). Taking
into account that the reactor operated at ambient temperature and no
energy was used for its heating, 80% of the consumed energy seems to be
Table 2
Energy balance in the AnMBBR treating medium-strength dairy wastewater (No
heating of the AnMBBR was applied in these experiments, the temperature in the
reactor was equal to 18 ± 2 ◦ C).
P Energy value (kWh/m3)
Energy Production
Electricity 0.221
Heat 0.316
Total 0.538
Energy consumption
Pumping 0.005
Mixing 0.020
Heating 0.000 (T:18 ◦ C) 0.232 (T:17 ◦ C) 0.464 (T:16 ◦ C)
Total 0.025 0.257 0.489
Energy Balance 0.513 0.281 0.049
*T = influent temperature.
E. Zkeri et al. Bioresource Technology 323 (2021) 124629

required for liquor’s mixing while the rest for wastewater’s pumping. It
is mentioned that the energy consumption increased significantly in case
of influent heating. For example, in areas with a mean annual temper­
ature of 17 ◦ C or 16 ◦ C the energy consumption increased to 0.257 kWh/
m3 and 0.489 kWh/m3, respectively (Table 2). Therefore, the operation
of the AnMBBR with medium-strength dairy wastewater seems more
favorable in tropical, sub-tropical and some Mediterranean regions.
There is very little knowledge about the energy recovery potential of
AnMBBR systems. Gu et al. (2017) estimated an electricity production of
0.191 kWh m− 3 from an AnMBBR treating domestic wastewater at a
temperature of 30 ◦ C. This value is slightly lower comparing with the
findings in this study (0.221 kWh m− 3). It is mentioned that data about
the energy required for heating the experimental appliance was not
reported in that work. Another previous work (Mei et al., 2016)
regarding the use of anaerobic membrane bioreactor (AnMBR) found an
energy production ranged from 0.17 kWh to 0.42 kWh per m3 of do­
mestic wastewater depending on reactor temperature (15–35 ◦ C). In
addition, low energy requirements for pumping (0.006 kWh m− 3) and
mixing (0.006–0.012 kWh m− 3) were reported, similar to this work. On
the other hand, Chen et al. (2019) estimated a net energy potential of
35.9 kWh m− 3 from an AnMBR treating a high strength influent (45.88 g
COD L− 1) from coffee processing wastewater and waste activated
sludge. It is obvious that for low and medium strength wastewater such
as domestic wastewater and the examined dairy wastewater the amount
of biogas (or energy) produced per m3 of influent is quite low in com­
parison with biogas produced from high strength wastewater. As a
result, the large influent specific heat (1.16 kWh m− 3 ◦ C− 1) has a major
impact on net energy recovery. For example, the energy required to heat
the influent from 20 ◦ C to 30 ◦ C is 11.6 kWh m− 3. A novel heat-pump
technology could transfer heat energy from the digestate into the feed
wastewater reducing the pre-heating demand at 80% (Xiao et al., 2018).
However, the remaining 20% (2.32 kWh m− 3 in the example) is still
quite high in comparison with total energy produced from low strength
wastewater (around 0.5 kWh m− 3). For these reasons, the operation of
AnMBBR with low and medium strength wastewater seems feasible only
at ambient temperatures.
It should also be mentioned that the use of AnMBBR as an A-stage
system reduces the COD entering in the aerobic MBBR (B-stage) and as a
result the energy required for aeration in the second stage is lower. For
example, Gu et al. (2017) calculate an energy saving for aeration of 0.23
kWh m− 3 from the use of AnMBBR as a first stage process in comparison
with simple conventional activated sludge process. These savings should
also be taken into consideration on the overall energy efficiency of the
AnMBBR systems.
Fig. 2. Change of biomass concentration and pH values in batch experiments
with Chlorella sorokiniana and medium strength dairy wastewater. Experiments
lasted 7 days and the effect of different daily light duration was checked (24 h
per day light versus 16 h light – 8 h dark).
d− 1, respectively (Table S4). As the pH was not adjusted during the
experiments and CO2 was not added, pH values increased gradually to
10.84 ± 0.11 and 10.45 ± 0.11 (Fig. 2). Similar pH changes have also
reported in the literature and are due to the CO2 consumption by the
microalgae that affect the balance of inorganic carbon (Asadi et al.,
2019). When cultivated under conditions of 16 h light per day (Exper­
iment B), the acclimatized biomass had a specific growth rate higher by
18% as compared to the non-acclimatized (Table S4).
Concerning the removal of major pollutants in these experiments,
COD was almost totally removed under all tested experimental condi­
tions, exceeding 98%% up to the 7th day (Fig. 3, Table S4). The average
total removal of NH4-N was higher than 74% in all batch experiments
and no statistical differences were noticed. On the other hand, signifi­
cantly higher PO4-P removal was achieved when acclimatized biomass
was used reaching 58.6% up to the end of the experiment (Fig. 3). In a
recent study conducted with Chlorella sorokiniana strain pa.91 and pre­
treated dairy wastewater that presented similar COD concentration with
the current study but lower concentrations of nutrients, a removal of
COD, NH4-N and PO4-P equal to 89%, 94% and 85% respectively was
observed in a period of 7 days (Asadi et al., 2019).
Analysis of the microalgal biomass at the end of the Experiment A
showed that its protein content was not significantly affected by the light
duration ranging between 38.6 and 39.6%, while a higher percentage of

3.2. Growth of Chlorella sorokiniana to dairy wastewater

As reported in Paragraph 2.3, Chlorella sorokiniana was gradually

acclimatized to filtered dairy wastewater. After acclimatization, its 72-h
specific growth rate in the presence of 40% dairy wastewater was equal
to 0.42 ± 0.14 d− 1, while the relevant values for the microalgae grown
in medium and secondary treated municipal wastewater were 0.77 ±
0.07 d− 1 and 0.80 ± 0.11 d− 1, respectively. These results show that
Chlorella sorokiniana can be grown in dairy wastewater, beside the
partial decrease of its growth rate that is attributed to the dairy waste­
water characteristics.
In order to specify the optimal conditions for using Chlorella sor­
okiniana for dairy wastewater treatment, batch experiments were con­
ducted with effluents from the AnMBBR and the role of light duration as
well as biomass acclimatization was examined. No significant differ­
ences were observed on the concentrations of biomass during the
experiment as well as on the observed growth rates when different daily
light duration was applied. Specifically, the concentrations of biomass at
the end of the experiment (7 d) were equal to 321 ± 10 mg L− 1 (24 h Fig. 3. Removal of COD, NH4-N and PO4-P in the batch experiments conducted
light) and 317 ± 5 mg L− 1 (16 h light/8h dark) (Fig. 2), while the values with Chlorella sorokiniana and medium strength dairy wastewater (duration of
of 7-d specific growth rates were 0.198 ± 0.005 d− 1 and 0.197 ± 0.002 the experiments: 7 d).

5
E. Zkeri et al.
proteins (44%) was noticed in the experiment with acclimatized
biomass. Slightly higher starch (3.6%) and lipids content (27.9%) were
also measured when acclimatized biomass was used (Table S4). Taking
into account that, for the tested conditions, the duration of the daily
light did not affect the growth and characteristics of microalgae, as well
as the removal of pollutants, the application of 16 h light per day as well
as the use of acclimatized biomass were selected for the following
experiments.
3.3. Sequential use of AnMBBR and microalgae SBR reactor for dairy
wastewater treatment
To evaluate the combined use of the anaerobic MBBR with a
microalgae reactor for medium-strength dairy wastewater treatment,
effluents from the AnMMBR that operated under OLR values of 2.5 kg
COD m− 3 d− 1, were collected and used for the operation of the SBR with
Chlorella sorokiniana in sequential experimental cycles (duration of each
cycle: 3 d). The performance of the AnMBBR was similar to that
described in Paragraph 3.1 achieving on average 40% COD removal
with simultaneous biogas production. The removal of other pollutants
was of minor importance, not exceeding 10%.
The pH of the SBR influents was 8.12 ± 0.11, while a moderate in­
crease to 8.58 ± 0.38 was observed at the effluents (Table 3). Almost the
total of the residual COD was removed after the treatment with micro­
algae (98 ± 1), while NH4-N, TKN and PO4-P total removal reached 65
± 12%, 69 ± 11% and 31 ± 11%, respectively. As a result, the average
concentration of CODdis at the effluents was lower than 30 mg L− 1, while
the concentrations of other pollutants ranged between 13.3 mg L− 1
(PO4-P) and 24.1 mg L− 1 (TKN) (Table 3). No failure of the system or
deterioration of its performance due to predation by zooplankton or
invertebrates was noticed during the experiment.
Considering the experimental conditions applied and the fact than no
sterilization of wastewater was carried out, the co-existence of micro­
algae and bacteria seems to contribute to the removal of COD. It well-
known that Chlorella sorokiniana can assimilate organic carbon under
mixotrophic conditions (Lizzul et al., 2018), while important COD
removal has also observed under mixotrophic conditions in previous
studies with municipal (Kotoula et al., 2020) or dairy wastewater (Asadi
et al., 2019). An NH4-N removal rate of 12.8 ± 4.4 mg L− 1 d− 1 was
calculated in the current experiments. The assimilation of ammonium
nitrogen by microalgae seems to be the main mechanism responsible for
its removal in the applied experimental conditions. Additionally to this
mechanism, the increased pH values observed in this study indicate that
NH3 volatilization could also have contributed to NH4-N removal. P
removal rate was equal to 2.0 ± 0.7 mg/L/d. As the average pH in the
SBR was around 8.5, the assimilation of orthophosphates to microalgae
seems to be the major mechanism for P removal in these experiments;
removal due to precipitation seems to be of minor importance as it
Table 3
Characteristics of the wastewater and the microalgal biomass in the SBR reactor
with Chlorella sorokiniana (n = number of analyzed samples). The influent
wastewater had been subjected to anaerobic treatment with AnMBBR.
Wastewater Characteristics (n = 7)
Influents Effluents
T (oC) 21.6 ± 3.7 26.5 ± 2.1
pH 8.12 ± 0.11 8.58 ± 0.38
CODdis (mg L− 1) 1258 ± 459 26 ± 1
NH4-N (mg L− 1) 58.3 ± 11.0 19.9 ± 4.7
TKN (mg L− 1) 79.2 ± 14.1 24.1 ± 8.3
PO4-P (mg L− 1) 19.3 ± 3.2 13.3 ± 3.1
Biomass Characteristics (n = 1)
Start of the experiment End of the experiment
Total Proteins (%) 42.79 54.56
Starch (%) 2.68 3.39
Lipids (%) 39.1 23.1
6
Bioresource Technology 323 (2021) 124629
manly occurs at pH > 8.5 (Wang et al. 2017).
During the experiment, 3382 mg of microalgal biomass were pro­
duced in the SBR (70 mg L− 1 d− 1, μ equal to 0.10 d− 1). Analysis of
microalgal biomass at the start and end of the experiment showed an
increase of protein and starch content to 54.56% and 3.39%, respec­
tively and a parallel decrease of lipids concentration to 23.1% (Table 3).
It is widely accepted that the abundance of nutrients enhance biomass
productivity and results to high protein in the produced microalgal
biomass, while nutrient stress conditions enhance high lipid accumula­
tion (Singh et al., 2016). Concerning Chlorella sorokiniana, experiments
using synthetic medium as the growth substrate have also shown that
the increase of pH enhance proteins content (Qiu et al., 2017). Previous
studies with other types of agro industrial wastewater and Chlorella
sorokiniana have reported that the characteristics of the produced
biomass vary according to the type of the wastewater and the experi­
mental conditions. Singh et al (2011) used diluted poultry litter anaer­
obic digester effluent and observed a maximum daily biomass
productivity of 66 mg L− 1 d− 1 while the content of dried biomass was in
the range 37–39% protein, 12–22% carbohydrates and 4–16% lipids.
Kobayashi et al. (2013) reported a biomass productivity of 12.77 mg L− 1
d− 1 when Chlorella sorokiniana UTEX 1230 was cultivated in diluted
anaerobic digested effluent from cattle manure. The protein content of
the biomass was equal to 33.94%, while the starch and lipids content
was 22.76% and 26.83%, respectively. In experiments with aquaculture
wastewater, Ansari et al. (2017) reported a biomass productivity of
107.86 mg L− 1 d− 1, while the protein, carbohydrates and lipids were
equal to 28.8%, 35.4% and 31.9%, respectively.
3.4. Comparison of the different systems for medium strength dairy
wastewater treatment and future challenges
This study showed that AnMBBR can be used for the pretreatment of
medium strength dairy wastewater achieving partial COD removal and
biogas production. The energy balance calculations revealed also that
energy autonomy can be attained when working at ambient tempera­
tures. The coupling of this reactor with an AeMBBR can produce an
effluent with very low concentrations of NH4-N and TKN which however
requires a further polishing step to meet the COD and TP standards of the
EU 91/271 Directive for treated wastewater disposal to the aquatic
environment (EU, 1991). The combination of the AnMBBR with a
microalgae reactor can produce an effluent with very low concentration
of COD that can be used for agricultural irrigation due to the high
concentrations of contained N and P. The harvesting of microalgal
biomass and its use as animal feedstock due to the high protein content
could provide extra revenue to dairy industry. It should be noted that
previous articles have also proposed the use of Chlorella sorokiniana as
feed for aquacultures (Barone et al., 2018; Zhang et al., 2019). Specific
points that need optimization before applying these systems in full-scale
are the required pretreatment of the AnMMBR effluents before their
introduction to the microalgae reactor for removing suspended solids
and turbidity as well as the harvesting method that could be applied for
biomass collection. The main strong points, the observed limitations and
the needs for further research of the studied systems are summarized in
Table 4.
4. Conclusions
The combination of a methanogenic and an aerobic MBBR achieves
significant removal of COD (93%), NH4-N (97%) and TKN (99%) and
partial removal of PO4-P (49%). The coupling of methanogenic MBBR
with a microalgae reactor results to full elimination of COD but lower
removal of NH4-N (65%), TKN (69%) and PO4-P (31%). The methano­
genic MBBR can be used for the pretreatment of medium strength dairy
wastewater as it has stable performance and energy autonomy at
ambient temperatures. The protein content of microalgal biomass
exceeded 54% indicating its potential valorization as high protein
E. Zkeri et al. Bioresource Technology 323 (2021) 124629

Table 4
Strong points, limitations and requirements for further research on the studied systems.
Studied Systems Strong Points Limitations Future challenges Maturity of the technology

AnMBBR − Partial COD removal
− Biogas production
− No energy demand
AnMBBR - AeMBBR − High removal of NH4-N,
TKN
− High removal of COD
AnMMBR – − Full COD removal
Microalgae − Treated WW suitable for
Reactor irrigation
− Biomass valorization as
animal feedstock
− No removal of N, P
− Does not meet the limit values of
the legislation for COD and TP
− Energy demand for the aerobic
step
− Energy demand for microalgae
cultivation
− Operation at higher OLR and T
− Addition of an extra step for meeting COD,
TP requirements
− Method selection for harvesting of
biomass and for AnMBBR effluent
pretreatment
− Sufficient as the first step of
dairy WW treatment
− Operation in pilot-scale before
designing the full-scale system
− Operation in pilot-scale before
designing the full-scale system

feedstock. Environ. Sci. Pollut. Res. 26 (28), 29473–29489. https://doi.org/10.1007/s11356-

CRediT authorship contribution statement
Eirini Zkeri: Conceptualization, Investigation. Athanasia Iliopou­
lou: Investigation, Visualization. Alexandra Katsara: Investigation.
Angeliki Korda: Investigation. Maria Aloupi: Methodology, Valida­
tion, Writing - review & editing. Georgia Gatidou: Formal analysis,
Writing - original draft. Michail S. Fountoulakis: Methodology, Visu­
alization, Writing - review & editing. Athanasios S. Stasinakis: Su­
pervision, Writing - original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We acknowledge support of this work by the project «FoodOmicsGR
Comprehensive Characterisation of Foods» (MIS 5029057, http://foo­
domics.gr/) which is implemented under the Action “Reinforcement of
the Research and Innovation Infrastructure”, funded by the Operational
Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF
2014–2020) and co-financed by Greece and the European Union (Eu­
ropean Regional Development Fund). A.S. Stasinakis would like to thank
Cyprus University of Technology (Department of Chemical Engineering)
for hosting him in the period that this article was written.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2020.124629.
References
Ahmad, T., Aadil, R.M., Ahmed, H., Rahman, U.U., Soares, B.C.V., Souza, S.L.Q.,
Pimentel, T.C., Scudino, H., Guimarães, J.T., Esmerino, E.A., Freitas, M.Q.,
Almada, R.B., Vendramel, S.M.R., Silva, M.C., Cruz, A.G., 2019. Treatment and
utilization of dairy industrial waste: A review. Trends Food Sci. Technol. 88,
361–372. https://doi.org/10.1016/j.tifs.2019.04.003.
Andreottola, G., Foladori, P., Ragazzi, M., Villa, R., 2002. Dairy wastewater treatment in
a moving bed biofilm reactor. Water Sci. Technol. 45, 321–328. https://doi.org/
10.2166/wst.2002.0441.
APHA, 2005. Standard methods for the examination of water and wastewater, 21st ed.,
American Public Health Association, 2005, USA.
Ansari, F.A., Singh, P., Guldhe, A., Bux, F., 2017. Microalgal cultivation using
aquaculture wastewater: Integrated biomass generation and nutrient remediation.
Algal Res. 21, 169–177. https://doi.org/10.1016/j.algal.2016.11.015.
AOAC International, 2002. AOAC Official Method 2001.11: Protein (crude) in animal
feed, forage (plant tissue), grain and oilseeds, 2002 AOAC International.
Asadi, P., Rad, H.A., Qaderi, F., 2019. Comparison of Chlorella vulgaris and Chlorella
sorokiniana pa.91 in post treatment of dairy wastewater treatment plant effluents.
019-06051-8.
Barone, R.S.C., Sonoda, D.Y., Lorenz, E.K., Cyrino, J.E.P., 2018. Digestibility and pricing
of chlorella sorokiniana meal for use in tilapia feeds. Sci. Agric. 75 (3), 184–190.
https://doi.org/10.1590/1678-992x-2016-0457.
Buntner, D., Sánchez, A., Garrido, J.M., 2013. Feasibility of combined UASB and MBR
system in dairy wastewater treatment at ambient temperatures. Chem. Eng. J. 230,
475–481. https://doi.org/10.1016/j.cej.2013.06.043.
Carvalho, F., Prazeres, A.R., Rivas, J., 2013. Cheese whey wastewater: Characterization
and treatment. Sci. Total Environ. 445–446, 385–396. https://doi.org/10.1016/j.
scitotenv.2012.12.038.
Chen, R., Wen, W., Jiang, H., Lei, Z., Li, M. Li, Y-Y., 2019. Energy recovery potential of
thermophilic high-solids co-digestion of coffee processing wastewater and waste
activated sludge by anaerobic membrane bioreactor. Bioresour. Technol. 274, 127-
133. 10.1016/j.biortech.2018.11.080.
di Biase, A., Devlin, T.R., Kowalski, M.S., Oleszkiewicz, J.A., 2018. Performance and
design considerations for an anaerobic moving bed biofilm reactor treating brewery
wastewater: Impact of surface area loading rate and temperature. J. Environ.
Manage. 216, 392–398. https://doi.org/10.1016/j.jenvman.2017.05.093.
D’Oca, M.G.M., Viêgas, C.V., Lemões, J.S., Miyasaki, E.K., Morón-Villarreyes, J.A.,
Primel, E.G., Abreu, P.C., 2011. Production of FAMEs from several microalgal lipidic
extracts and direct transesterification of the Chlorella pyrenoidosa. Biomass Bioenergy
35 (4), 1533–1538. https://doi.org/10.1016/j.biombioe.2010.12.047.
EU, 1991. Council Directive 91/271/EEC of 21 May 1991 concerning urban waste-water
treatment. eur-lex.europa.eu/legalcontent/EN/TXT/HTML/?uri=CELEX:
31991L0271&from=EN (last access: 28.10.2020).
Gatidou, G., Anastopoulou, P., Aloupi, M., Stasinakis, A.S., 2019. Growth inhibition and
fate of benzotriazoles in Chlorella sorokiniana cultures. Sci. Total Environ. 663,
580–586. https://doi.org/10.1016/j.scitotenv.2019.01.384.
Goli, A., Shamiri, A., Khosroyar, S., Talaiekhozani, A., Sanaye, R., Azizi, K., 2019.
A review on different aerobic and anaerobic treatment methods in dairy industry
wastewater. J. Environ. Treat. Tech. 7, 113–141.
Gu, J., Xu, G., Liu, Y., 2017. An integrated AMBBR and IFAS-SBR process for municipal
wastewater treatment towards enhanced energy recovery, reduced energy
consumption and sludge production. Water Res. 110, 262–269. https://doi.org/
10.1016/j.watres.2016.12.031.
Gupta, S., Pawar, S.B., Pandey, R.A., 2019. Current practices and challenges in using
microalgae for treatment of nutrient rich wastewater from agro-based industries. Sci.
Total Environ. 687, 1107–1126. https://doi.org/10.1016/j.scitotenv.2019.06.115.
Hansen, J., Møller, I., 1975. Percolation of starch and soluble carbohydrates from plant
tissue for quantitative determination with anthrone. Anal. Biochem. 68 (1), 87–94.
https://doi.org/10.1016/0003-2697(75)90682-X.
Hena, S., Fatihah, N., Tabassum, S., Ismail, N., 2015. Three stage cultivation process of
facultative strain of Chlorella sorokiniana for treating dairy farm effluent and lipid
enhancement. Water Res. 80, 346–356. https://doi.org/10.1016/j.
watres.2015.05.001.
Kobayashi, N., Noel, E.A., Barnes, A., Watson, A., Rosenberg, J.N., Erickson, G., Oyler, G.
A., 2013. Characterization of three Chlorella sorokiniana strains in anaerobic digested
effluent from cattle manure. Bioresour. Technol. 150, 377–386. https://doi.org/
10.1016/j.biortech.2013.10.032.
Kora, E., Theodorelou, D., Gatidou, G., Fountoulakis, M.S., Stasinakis, A.S., 2020.
Removal of polar micropollutants from domestic wastewater using a methanogenic –
aerobic moving bed biofilm reactor system. Chem. Eng. J. 382, 122983. https://doi.
org/10.1016/j.cej.2019.122983.
Kotoula, D., Iliopoulou, A., Irakleous-Palaiologou, E., Gatidou, G., Aloupi, M.,
Antonopoulou, P., Fountoulakis, M.S., Stasinakis, A.S., 2020. Municipal wastewater
treatment by combining in series microalgae Chlorella sorokiniana and macrophyte
Lemna minor: Preliminary results. J. Clean. Prod. 271, 122704. https://doi.org/
10.1016/j.jclepro.2020.122704.
Lantz, M., 2012. The economic performance of combined heat and power from biogas
produced from manure in Sweden - a comparison of different CHP technologies.
Appl. Energy 98, 502–511. https://doi.org/10.1016/j.apenergy.2012.04.015.
Lizzul, A.M., Lekuona-Amundarain, A., Purton, S., Campos, L.C., 2018. Characterization
of Chlorella sorokiniana, UTEX 1230. Biology 7, 25. https://doi.org/10.3390/
biology7020025.

7
E. Zkeri et al.
Luostarinen, S.A., Rintala, J.A., 2005. Anaerobic on-site treatment of black water and
dairy parlour wastewater in UASB-septic tanks at low temperatures. Water Res. 39
(2-3), 436–448. https://doi.org/10.1016/j.watres.2004.10.006.
Luostarinen, S., Luste, S., Valentín, L., Rintala, J., 2006. Nitrogen removal from on-site
treated anaerobic effluents using intermittently aerated moving bed biofilm reactors
at low temperatures. Water Res. 40 (8), 1607–1615. https://doi.org/10.1016/j.
watres.2006.02.022.
Marshall, J.D., 1986. Drought and shade interact to cause fine-root mortality in Douglas-
fir seedlings. Plant Soil 91 (1), 51–60. https://doi.org/10.1007/BF02181818.
Mamais, D., Noutsopoulos, C., Dimopoulou, A., Stasinakis, A., Lekkas, T.D., 2015.
Wastewater treatment process impact on energy savings and greenhouse gas
emissions. Water Sci. Technol. 71, 303–308. https://doi.org/10.2166/wst.2014.521.
Mazioti, A.A., Stasinakis, A.S., Pantazi, Y., Andersen, H.R., 2015. Biodegradation of
benzotriazoles and hydroxy-benzothiazole in wastewater by activated sludge and
moving bed biofilm reactor systems. Bioresour. Technol. 192, 627–635. https://doi.
org/10.1016/j.biortech.2015.06.035.
Mei, X., Wang, Z., Miao, Y., Wu, Z., 2016. Recover energy from domestic wastewater
using anaerobic membrane bioreactor: operating parameters optimization and
energy balance analysis. Energy 98, 146–154. https://doi.org/10.1016/j.
energy.2016.01.011.
Meister, M., Rezavand, M., Ebner, C., Pümpel, T., Rauch, W., 2018. Mixing non-
Newtonian flows in anaerobic digesters by impellers and pumped recirculation. Adv.
Engin. Softw. 115, 194–203. https://doi.org/10.1016/j.advengsoft.2017.09.015.
Mohsenpour, S.F., Hennige, S., Willoughby, N., Adeloye, A., Gutierrez, T., 2021.
Integrating micro-algae into wastewater treatment: A review. Sci. Total Environ.
752, 142168. https://doi.org/10.1016/j.scitotenv.2020.142168.
Nagarajan, D., Lee, D.-J., Chen, C.-Y., Chang, J.-S., 2020. Resource recovery from
wastewaters using microalgae-based approaches: A circular bioeconomy perspective.
Bioresour. Technol. 302, 122817. https://doi.org/10.1016/j.biortech.2020.122817.
OECD, 2011. Guidelines for the testing of chemicals, Section 2, Test No. 201: Freshwater
Alga and Cyanobacteria, Growth Inhibition Test, pp 1-25 (https://www.oecd-
ilibrary.org/environment/test-no-201-alga-growth-inhibition-test_9789264069923-
en, last access 21.10.2020).
Pandey, A., Srivastava, S., Kumar, S., 2019. Isolation, screening and comprehensive
characterization of candidate microalgae for biofuel feedstock production and dairy
effluent treatment: A sustainable approach. Bioresour. Technol. 293, 121998.
https://doi.org/10.1016/j.biortech.2019.121998.
Qiu, R., Gao, S., Lopez, P.A., Ogden, K.L., 2017. Effects of pH on cell growth, lipid
production and CO2 addition of microalgae Chlorella sorokiniana. Algal Res. 28,
192–199. https://doi.org/10.1016/j.algal.2017.11.004.
Ruggeri, B., Tommasi, T., Sassi, G., 2010. Energy balance of dark anaerobic fermentation
as a tool for sustainability analysis. Int. J. Hydr. Energy 35 (19), 10202–10211.
https://doi.org/10.1016/j.ijhydene.2010.08.014.
Singh, M., Reynolds, D.L., Das, K.C., 2011. Microalgal system for treatment of effluent
from poultry litter anaerobic digestion. Bioresour. Technol. 102 (23), 10841–10848.
https://doi.org/10.1016/j.biortech.2011.09.037.
Bioresource Technology 323 (2021) 124629
Singh, P., Kumari, S., Guldhe, A., Misra, R., Rawat, I., Bux, F., 2016. Trends and novel
strategies for enhancing lipid accumulation and quality in microalgae. Renew. Sust.
Energ. Rev. 55, 1–16. https://doi.org/10.1016/j.rser.2015.11.001.
Slavov, A.K., 2017. General characteristics and treatment possibilities of dairy
wastewater -a review. Food Technol. Biotechnol. 55, 14–28. https://doi.org/
10.17113/ftb.55.01.17.4520.
Tawfik, A., Sobhey, M., Badawy, M., 2008. Treatment of a combined dairy and domestic
wastewater in an up-flow anaerobic sludge blanket (UASB) reactor followed by
activated sludge (AS system). Desalination 227 (1-3), 167–177. https://doi.org/
10.1016/j.desal.2007.06.023.
Thiex N. J., Manson H., Anderson S., Persson J.A., 2002. Determination of crude protein
in animal feed, forage, grain, and oilseeds by using block digestion with a copper
catalyst and steam distillation into boric acid: collaborative study. J. AOAC Int. 85,
309-317. 10.1093/jaoac/85.2.309.
Torresi, E., Fowler, S.J., Polesel, F., Bester, K., Andersen, H.R., Smets, B.F., Plósz, B.G.,
Christensson, M., 2016. Biofilm thickness influences biodiversity in nitrifying MBBRs
- Implications on micropollutant removal. Environ. Sci. Technol. 50 (17),
9279–9288. https://doi.org/10.1021/acs.est.6b0200710.1021/acs.est.6b02007.
s001.
Vyrides, I., Drakou, E.M., Ioannou, S., Michael, F., Gatidou, G., Stasinakis, A., 2018.
Biodegradation of bilge water: batch test under anaerobic and aerobic conditions
and performance of three pilot Aerobic Moving Bed Biofilm Reactors (MBBRs) at
different filling fractions. J. Environm. Manage. 217, 356–362. https://doi.org/
10.1016/j.jenvman.2018.03.086.
Xiao, B., Qin, Y., Wu, J., Chen, H., Yu, P., Liu, J., Li, Y.Y., 2018. Comparison of single-
stage and two-stage thermophilic anaerobic digestion of food waste: performance,
energy balance and reaction process. Energy Convers. Manag. 156, 215–223.
https://doi.org/10.1016/j.enconman.2017.10.092.
Zhu, S., Feng, S., Xu, Z., Qin, L., Shang, C., Feng, P., Wang, Z., Yuan, Z., 2019. Cultivation
of Chlorella vulgaris on unsterilized dairy-derived liquid digestate for simultaneous
biofuels feedstock production and pollutant removal. Bioresour. Technol. 285,
121353. https://doi.org/10.1016/j.biortech.2019.121353.
Wang, J.-H., Zhang, T.-Y., Dao, G.-H., Xu, X.-Q., Wang, X.-X., Hu, H.-Y., 2017.
Microalgae-based advanced municipal wastewater treatment for reuse in water
bodies. Appl. Microbiol. Biotechnol. 101 (7), 2659–2675. https://doi.org/10.1007/
s00253-017-8184-x.
Wang, Y., Serventi, L., 2019. Sustainability of dairy and soy processing: A review on
wastewater recycling. J. Clean. Prod. 237, 117821. https://doi.org/10.1016/j.
jclepro.2019.117821.
Zhang, L., Pei, H., Yang, Z., Wang, X., Chen, S., Li, Y., Xie, Z., 2019. Microalgae nourished
by mariculture wastewater aids aquaculture self-reliance with desirable biochemical
composition. Bioresour. Technol. 278, 205–213. https://doi.org/10.1016/j.
biortech.2019.01.066.