J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 3 7–5 0

Available online at www.sciencedirect.com

ScienceDirect
www.journals.elsevier.com/journal-of-environmental-sciences

Investigation on removal pathways of Di 2-ethyl hexyl

phthalate from synthetic municipal wastewater using a
submerged membrane bioreactor

Mehdi Zolfaghari1 , Patrick Drogui1,⁎, Brahima Seyhi1 , Satinder Kaur Brar1 , Gerardo Buelna2 ,
Rino Dubé2 , Nouha Klai1
1. National Institute of Scientific Research, Water, Earth and Environment (INRS-ETE), University of Quebec, Quebec City,
Quebec G1K 9A9, Canada
2. Industrial Research Center of Quebec, Quebec City, Quebec G1P 4C7, Canada

AR TIC LE I N FO ABS TR ACT

Article history: Highly hydrophobic Di 2-ethyl hexyl phthalate (DEHP) is one of the most prevalent
Received 8 January 2015 plasticizers in wastewaters. Since its half-life in biological treatment is around 25 days, it
Revised 19 February 2015 can be used as an efficiency indicator of wastewater treatment plant for the removal of
Accepted 5 March 2015 hydrophobic emerging contaminants. In this study, the performance of submerged
Available online 2 July 2015 membrane bioreactor was monitored to understand the effect of DEHP on the growth
of aerobic microorganisms. The data showed that the chemical oxygen demand (COD)
Keywords: and ammonia concentration were detected below 10 and 1.0 mg/L, respectively for
Di 2-ethyl hexyl phthalate operating conditions of hydraulic retention time (HRT) = 4 and 6 hr, sludge retention
Submerged membrane bioreactor time (SRT) = 140 day and sludge concentration between 11.5 and 15.8 g volatile solid
Removal efficiency (VS)/L. The removal efficiency of DEHP under these conditions was higher and ranged
Soluble microbial products between 91% and 98%. Results also showed that the removal efficiency of DEHP in
Toxicity biological treatment depended on the concentration of sludge, as adsorption is the main
mechanism of its removal. For the submerged membrane bioreactor, the pore size is the
pivotal factor for DEHP removal, since it determines the amount of soluble microbial products
coming out of the process. Highly assimilated microorganisms increase the biodegradation
rate, as 74% of inlet DEHP was biodegraded; however, the concentration of DEHP inside sludge
was beyond the discharge limit. Understanding the fate of DEHP in membrane bioreactor,
which is one of the most promising and futuristic treatment process could provide
replacement for conventional processes to satisfy the future stricter regulations on emerging
contaminants.
© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences.
Published by Elsevier B.V.

Introduction growth and reproduction hormones, especially at early age.

Therefore, they are known as potential endocrine distrusting
The structural similarities between phthalate esters and compounds (Tan et al., 2007), already controlled and banned
endogenous estrogens may result in interruption of human by European, North American, and east Asian countries (Sun

⁎ Corresponding author. E-mail: patrick.drogui@ete.inrs.ca (Patrick Drogui).

http://dx.doi.org/10.1016/j.jes.2015.03.025
1001-0742 © 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
38 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 37–5 0
et al., 2014). Around the world, especially in Europe, Di 2-ethyl
hexyl phthalate (DEHP) has also been considered as the most
detectable plasticizer and one of the most problematic
emerging contaminants (Zolfaghari et al., 2014). In Canada,
DEHP was accounted as the highest non-steroidal synthetic
compounds in both municipal influent and effluent samples
(Fernandez et al., 2007).
Frequent detection of these compounds in environment,
typically near municipal areas represent its anthropogenic
sources, which are closely connected with the continuous
discharge of trace concentrations by treated wastewater. For
instance, DEHP concentration increased from an average of
150 μg/L in upstream to 171 μg/L in river downstream, due to
constant release of 162 μg/L by wastewater treatment plant
(WWTP) effluent (Olujimi et al., 2012). Another example in the
province of Quebec showed constant release of 54 μg/L of DEHP
from Montreal WWTP effluent into Saint-Lawrence River raised
its concentration to 180 μg/L in running water. Conventional
physicochemical processes in Montreal WWTP are reported to
remove only 23% of inlet DEHP (Barnabé et al., 2008).
As a highly hydrophobic compound (logKow = 7.5) and its
non-volatility properties (henry constant low = 1.74–4.43
(Pa·m3)/mol), it mainly adsorbed to suspended solid and
macromolecules and rarely volatilized into the air. By gradual
degradation of its carrier, the major part of DEHP remains in
water or adsorbed onto soil and sediment. The rate of natural
biodegradation and physicochemical (e.g., photolysis and
hydrolysis) degradation of DEHP in aqueous system was so
slow, as its half-life is estimated in the range of 1–4.5 years
(Magdouli et al., 2013). Free DEHP molecules, stripping into the
air are vulnerable to photodegradation (Peterson and Staples,
2003). In solid phase, the degradation pace in sediment is
faster than soil probably due to the presence of waterborne
microorganisms and abundant oxygen. O'Grady et al. (1985)
estimated the rate of removal of DEHP in activated sludge. For
initial concentration of 3.3 mg DEHP/L, removal efficiency
reached 71% and 91% in 24 and 48 hr, respectively. By
considering the path of DEHP biodegradation, ether cleavage
and production of alcohol and phenol derivatives, such as
MEHP, di benzoic acid, and benzoic acid are the basic products
of degradation (Magdouli et al., 2013). The hydrolysis of each
chemical bond is more prone to electrophilic attack by oxygen
of aerobic bacteria (Alturki, 2013; Tadkaew et al., 2011). As a
consequence, the removal efficiencies of DEHP (electron donat-
ing group) are higher than anaerobic and anoxic processes.
Similar results were obtained by Huang et al. (2008) who showed
the removal efficiency proportion percentages of anaerobic,
anoxic and aerobic reactors to be in the range of 14%–23%, 15%–
19%, and 61%–68%, respectively.
Diverse kinds of plastic goods used by citizens, level of
municipality, discharge of landfill leachate and industrial
wastewaters into municipal wastewater and the method used
by researchers substantially determine the concentration of
DEHP. Levels of DEHP in municipal wastewater are widely
fluctuated between the range of 0.716 (Tan et al., 2007) to
379 μg/L (Camacho-Muñoz et al., 2014), and even at 1.085 mg/L
(Olujimi et al., 2012).
Recent studies mainly focus on the introduction of efficient
system for emerging contaminant removal or optimization and
modify the current systems, since slight change in operating
condition affected the efficiency of treatment plants for hydro-
philic compounds (Zolfaghari et al., 2014). Modification of
conventional activated sludge could be the ideal option for
enhancing the removal efficiency of hydrophobic compound,
since membrane rejection of suspended solids and sludge
diminished the emergence of lipophilic molecules owing to
size exclusion (Clara et al., 2005). In all former studies, adsorption
on suspended solids and/or mixed liquor suspended solid (MLSS)
has been reported as the main removal mechanism of DEHP.
Therefore, the concentration of DEHP sludge in WWTP reached
153 mg/kg, while the influent and effluent concentrations were
1.6 and 0.03 μg DEHP/L, respectively (Peterson and Staples, 2003).
Adsorbed DEHP onto activated sludge later biodegraded, espe-
cially under high sludge age condition (Boonyaroj et al., 2012b).
Therefore, increasing the age of sludge in membrane bioreactor
(MBR) provided sufficient biodegradation time.
In this study the performance of submerged membrane
bioreactor (SMBR) was closely studied to understand the effect
of DEHP on the performance parameters, such as chemical
oxygen demand (COD), NH4-nitrogen, and PO4-phosphorous
removal. Synthetic wastewater used not only stimulated the
municipal wastewater concentration, but also removed all
mediating factors that may affect tracing of DEHP fate in the
outlet. Furthermore, the ability of SMBR for removal enhance-
ment was investigated by mass balance DEHP.
1. Material and methods
1.1. Experimental unit
A laboratory scale SMBR consisting of a aeration basin with an
active volume of 4 L, a continuous mixer, air diffuser, a pressure
gage, and influent and effluent pumps was employed. The
stirred tank with 19.1 cm of water height was equipped with
Zeed-Weed (ZW-1) hollow-fiber membrane manufactured by
Zenon Environmental Inc. (Oakville, ON, Canada). It has 80
vertical fibers of 20 cm with the 0.04 μm pore diameter which
provided 0.047 m2 of total surface area for filtration. A pulsed
peristaltic pump produced vacuum inside the membrane. In
order to minimize the accumulation of sludge on the membrane
surface (prevent fouling), the pump operated intermittently in
cycle of 60/20 sec of filtration/rest. Furthermore, the extra air
supply went right through membrane for cake removal. Besides,
the negative pressure was controlled by manometer located on
the membrane aspiration line. The fouling of membrane was
constantly checked by trans-membrane pressure (TMP). For
TMP less than −45 kPa, backwashing was carried out by the
membrane permeate, while reaching TMP more than −69 kPa
(the highest operation pressure) membrane chemical wash was
performed. Firstly, membrane was washed by tap water to
remove the cake layer and then, it immersed in a solution of
NaOCl (1000 mg/L) for 2 hr. Strong oxidizing property of ClO−
removed the organic matters stuck in the membrane pores.
Acidic cleaning by 3.0 g/L of citric acid solution for 30 min was
sometimes performed for the removal of mineral clogged in the
membrane pores.
Air flow was controlled for keeping the dissolved oxygen
always more than 2 mg O2/L. Even though mixing was
provided by aeration, a variable speed mixer operated at
 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 3 7–5 0
600 r/min, using three-blade, stainless steel axial impellers
with 7 cm in diameter was used for further homogenization of
the aeration basin. The range of 18 ± 2°C and 7.2 ± 0.5 was
measured for the operating temperature and pH, respectively.
The use of ultrafiltration kept the amount of turbidity always
below 0.2 NTU during the course of experiment.
1.2. Chemicals
All the organic solvents including methanol (MeOH), acetone,
dichloromethane (DCM) were of analytical grade. DEHP, glu-
cose, (NH4)2SO4, KH2PO4, as well as all other minerals, used for
synthetic wastewater, were purchased from Sigma-Aldrich
Canada Ltd. (Oakville, ON, Canada).
1.3. Synthetic wastewater
Synthetic wastewater in this study was formulated to
simulate municipal sewage effluent. The exact compositions
of the synthetic wastewater as derived from the average
concentration of municipal wastewater in Canada are as
following (mg/L): glucose 400, DEHP 0.15, (NH4)2SO4 245.40,
KH2PO4 52.73, MgSO4 22.79, CaCl2·2H2O 14.67, FeCl3 7.73,
Na2CO3 304.17, CuSO4 0.05, Na2MoO4·2H2O 0.06, MnSO4·H2O
0.09, ZnCl2 0.17, and CoSO4·7H2O 0.31. The COD, nitrogen,
phosphorous and DEHP concentrations were in the same
range with average municipal wastewater. The average values
of COD, N-NH+4 and P-PO3+ 4 were around 440, 25 and 5 mg/L
with the C/N/P of 100/5/1. Because of its insolubility, DEHP was
dissolved in methanol (10 g/L). Feed solution was prepared
daily by mixing 500 mL of mineral solution containing
ammonia and phosphorous, 600 μL of DEHP stock solution,
8 g of glucose, and 19.5 L of distillated water.
1.4. Experimental procedure
For the start-up, the MBR was inoculated with activated
sludge used previously for the investigation of Bisphenol A
removal in MBR (Seyhi et al., 2012). The pilot reactor was run
for 40 days in the batch mode until the volatile solids (VS)
increase to 15 g/L. Later on, a small portion of sludge was weekly
extracted from the reactor for both total solid (TS) and VS
analysis, and maintaining the age of the sludge at approximately
140 days. The hydraulic retention time (HRT) was set at 8 hr,
corresponding to a permeate flux of 10.64 L/(m2·hr) (or flow of
8.3 mL/min). Afterward, the HRT of 6 hr was imposed to be in the
range of conventional wastewater treatment plants (4–8 hr)
(Seyhi et al., 2012), as well as increase the organic loading rate
into the range of 115 ± 10 mg COD/(g VS·day). Once the bioreactor
was stabilized for all the operation parameters, DEHP was
gradually introduced into the reactor (assimilation phase) from
the concentration of 20 to 150 μg/L, to be in the range of DEHP
concentration in municipal wastewater (Zolfaghari et al., 2014),
while the total COD was maintained around 400 mg/L. During
the course of three months, the biomass was acclimatized using
DEHP as a source of carbon. The operating conditions of the
SMBR plant during the experiment were as follows: HRT 4, 6, 8 hr;
membrane flux 10.64, 14.18, 21.28 L/(m2·hr); TS 5.09–18.07 g/L; VS
4.68–15.8 g/L, dissolved oxygen (DO) 3.1 ± 0.5 mg/L, sludge reten-
tion time (SRT) 140 days and temperature 18 ± 1.5°C.
39
For the batch adsorption test, the sludge taken from aeration
basin was fostered intensely to increase its concentration up to
10 g TS/L. The usual shaking erlenmeyer method was not
viable, as residual soluble microbial products (SMPs) interact
with dichloromethane (DCM), producing dense surfactant
which interrupt extraction. By utilization of two parallel
membranes, 200 mL was filtered out from the 20 L tank for
each sample in different times. Besides, 5 mg HgSO4/L was used
for deactivating the sludge in second phase of batch test.
1.5. Sampling
For performance inspection, sampling from influent and
effluent was performed twice per week. Around 1 L was taken
in glass bottle for extraction, frozen at −8°C before extraction, as
well as three 50 mL in plastic pipe for COD, nitrogen and
phosphorous test, were acidified and then stored at 4°C before
analysis. About 200 mL of reactor-mixed liquor sample was
taken from the aeration basin to determine total and volatile
solids, once every week.
1.6. Analytical methods
1.6.1. General parameters
According to the recommended standard methods, all of the
sample parameters, such as COD, VS, N-NH+4, N-NOx, P-PO3− 4
were frequently measured during the experimental span. In
conformity of standard method (MA. 315-DCO 1.0) proposed
by Center of Expertise in Environmental Analysis of Quebec,
COD was measured by colorimetric analysis in the presence of
potassium dichromate and the absorbance was measured at
600 nm using a UV spectrometer (Cary 50, Varian Canada Inc.,
Mississauga, ON, Canada). Ammonia nitrogen (NH3-N) and
phosphorus (PO4-P) were analyzed simultaneously according
to the analytical method proposed LACHAT Instrument
(QuikChem Method 10-107-06-2-B and 10-115-01-1-B).
1.6.2. Extraction of SMP
Around 80 mL of outlet reserved in 4°C for extraction of
extracellular polymeric substances (EPS), especially soluble
microbial product (SMP). Samples were centrifuged at 6000 g,
4°C for 20 min to obtain supernatant (containing Slime EPS,
termed as SMP) and the biomass pellet was re-suspended in
deionized water to initial volume (containing Capsular EPS,
termed as C-EPS) (Subramanian et al., 2010). To determine dry
weight of SMP, the supernatant and ethanol (95%, V/V) were
mixed in 1:2.2 ratios and kept for overnight at − 20°C to
precipitate it. The precipitated SMP was collected (as pellet)
by centrifugation (6000 g, 4°C, 20 min). Dry weight of the
pellet corresponding to the SMP concentration was measured
by drying the precipitates at 60°C to a constant weight
(A.P.H.A., 2005).
1.6.3. DEHP extraction
The aquatic samples were frozen at − 8°C before liquid-liquid
extraction. Glass was selected as the material for sampling
devices and laboratory equipment to avoid interferences of
plastic contamination. DEHP was extracted from 1 L of
matrices with 200 mL of DCM by means of Separatory funnel.
The extraction procedure was repeated twice by hardly
40 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 37–5 0
hand-shaking of 1 L sample and 100 mL of DCM. Later on, the
extracts were reduced from 200 to 4 mL by means of rotary
evaporator and then placed in two 1-mL vials for gas
chromatography (GC) analysis. Subsequently, n-pentadecane
was added into every vial as an internal standard. The
recovery of the extraction procedure was evaluated by
spiking known concentrations of DEHP into distillated water
and synthetic wastewater. The recovery amount for inlet and
outlet samples was 71%, and 78%, respectively.
For the sludge samples, the supernatant of SMBR firstly
gathered in aluminum container and dried in the oven with the
temperature of 45°C, since DEHP is a non-volatile compound.
Later diatomaceous earth was added to dry sludge by the
portion of 1:2 as a dispersing agent and thoroughly mixed by
mortar and pestle for homogenizing. Around 1.5 g of solid later
packed into accelerated solvent extractor (ASE) cell. 50:50 (V/V)
of dichloromethane:acetone at 110°C under 1500 psi pressure
used in the ASE device. The extraction procedure was adopted
in accordance with Reid et al. (2009). The solvent was later
evaporated by nitrogen gas in room temperature, dissolved by
DCM and then placed in two 1-mL vials.
1.6.4. GC/MS
For measurement of DEHP concentration, gas chromatography/
mass spectrometry (GC/MS) analysis was applied on a GC/MS
Clarus 500 (Perkin Elmer, Waltham, Massachusetts, USA) and a
VF-5 MS capillary column (Varian), 30 m long, 0.25 mm in
diameter and 0.25 μm film thickness. The MS detecting range
was 45 and 300 m/z. Helium was selected as the carrier gas at a
flow rate of 1 mL/min with the injection temperature of 280°C.
The ratio of 10:1 was selected for the injection of 1 μL of sample
into the column. The column temperature was initially held at
80 ± 1°C for 1 min, ramped to 280°C at a rate of 12°C/min, raised
again to 300°C by the rate of 15°C/min, then temperature was
held at 300°C for 1 min. Between four distinctive fragrances in
the mass spectrometer (71, 149, 167 and 279 m/z), 167 m/z was
selected as the reference peak, while 71 m/z fragment used for
calibration, as N-Pentadecane has the same peak. Quadratic
regression of peak area of standard samples against their
concentration was used for the calibration curves.
1.7. Toxicity test
The quality of synthetic and treated wastewater have been
measured by two set of biotest battery (Microtox and Daphnia
bioassay tests), to determine the acute toxicity of samples.
During microtox test, the luminescent marine, Vibrio fisheri,
was exposed to varying percentages of the samples, diluted
with reconstituted hard water (1.5%, 3.0%, 6.25%, 12.5%, 25.0%,
and 50%, V/V). In the first 10 min before adding sample, the
bioluminescence emitted by V. fisheri was measured after
incubation (control assay). After the addition of wastewater
samples (treated or untreated wastewater) into bacterial
suspension, the bioluminescence reduction was determined
after 5, 15 and 30 min exposure to the contaminant (Software
MTX6, version 6.0, Microbics Corporation, Shawnee, Kansas,
USA). The average percentage of light emission inhibition of V.
fisheri in comparison with the control assay was considered as
the toxicity effect of samples (Environnement Canada, 1992;
USEPA, 1993). The second test, Daphnia bioassay, was based on
the lethal concentration of sample in which at least 50% of
mortality of crustacean Daphnia magna was observed after
exposure time of 48 hr. The mortality rate of one control and
five serial dilutions of each sample (6.25%, 12.5%, 25.0%, 50.0%,
and 100%, V/V) were calculated by counting survived organisms
after 48 hr (LC50, 48 hr). Finally, the toxicity effect was evaluated
using statistic calculation software (Computer Basic, Spear-
man Karber tests, version 2.01, Microsoft, U.S. Environmental
Protection Agency, Cincinnati, Ohio, USA) (Environnement
Canada, 2000; Ministère de l'environnement du Québec,
2000).
2. Results and discussion
2.1. Reactor start-up
In order to develop the adequate concentration of microbial
consortia, the initial batch was performed. During this
operation (sequence batch reactor), 2 L of synthetic wastewa-
ter was introduced into the reactor every 12 hr, which
resulted in an increase of the concentration of microorgan-
isms to 17.2 g VS/L. Within this period, more than 90% of COD
and 99% of NH4-N were respectively removed. After charac-
terization of membrane for final sludge concentration, the
assimilation period was started by the initial concentration of
20 μg DEHP/L and HRT of 8 hr in continuous mode operation
(days 1 to 16). Later on, HRT was decreased to 6 hr and kept
constant until the end of assimilation period. In order to
prevent the sludge poisoning, the DEHP concentration was
gradually increased from 20 (16–40 days) to 50 (41–58 days),
100 (59–81 days) and at last 150 μg/L (82–92 days). It is worth
noting that the addition of DEHP took place whenever the
steady state condition reached and no further fluctuation in
SMBR performance was observed. Table 1 shows the MBR
performance during the acclimation period.
2.2. MBR performance
Desirable culture of microorganisms was well developed after
four months of operation, ensuring the realistic data on MBR
performance for DEHP removal in the continuous experiment.
HRT and concentration of sludge are among the primary
factors in MBR design, as they determine the removal
efficiency, energy consumption, volume and fouling of
membrane. HRT must be chosen as a balance between cost
and removal efficiency, since it directly affects the membrane
fouling and sludge performance (Viero and Sant'Anna, 2008).
Accordingly, three different HRTs (4, 6, and 8 hr) and two TS
range (14 and 7 g/L in average) were examined for estimation
of their effect on MBR parameters.
2.2.1. Sludge characteristics
Continuous biodegradable COD provided by glucose inside
the wastewater stimulated the growth of sludge in SMBR.
According to Fig. 1, at the beginning of continuous operation
the sludge concentration gradually declined as it accumulated
on the dead column zone of aeration tank. It seems that DEHP
gradually built up in the sludge mass; finally affecting the
toxicity of the specific range of microorganisms, expelling
 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 3 7–5 0 41

Table 1 – Basic performance of membrane bioreactor (MBR) during acclimation period.

Parameter Amount

Estimated DEHP load (mg/(L·day)) 0.08 0.2 0.4 0.6

Ratio (g COD/(g VS·day)) 0.106–0.144 0.116–0.126 0.142–0.154 0.135–0.149
COD removal (%) 92.2–99.2 93.4–97.7 98.6–85.6 97.4–99.8
DEHP removal (%) Around 100 62.4–88.6 67.7–97.8 96.4–98.4
NH4 removal (%) 91.4–99.8 99.1–99.4 97.7–100 83.8–100
PO4 removal (%) 0–92.8 76.5–96.2 24.6–64.4 0–36.7
TN removal (%) 0.7–95 74.1–88.7 75.4–98.4 0.1–35.1

DEHP: Di 2-ethyl hexyl phthalate, F/M: food/microorganism; VS: volatile solid; COD: chemical oxygen demand; TN: total nitrogen.

them from aeration basin. The sudden growth of sludge was
observed after the increase of organic load from 1.6 to 1.99 g
COD/(L·day) (shifting HRT from 8 to 6 hr). The analysis of
sludge characteristics during continuous phase showed that
the concentration of sludge became stable at 11.5 ± 0.2 g VS/L
by organic loading rate (OLR) of 1.77 ± 0.03 g COD/(L·day),
while decreasing the HRT from 6 to 4 hr, resulting in increase
in OLR to 1.97 ± 0.07 g COD/(L·day) and consequently linearly
triggered the growth of microorganisms. It seems that the
microorganisms tried to revive the previous food/microorgan-
ism (F/M) ratio by their growth; yet 14% increase of VS in
1 month finally resulted in membrane clogging and halting
the system in this HRT. It seems that the membrane fouling was
directly linked to F/M ratio and membrane flux (Trussell et al.,
2006). Rapid change of F/M ratio (0.168 to 0.418 g COD/(g VS·day))
on day 136, also triggered the growth of microorganisms. It
seems that F/M ratio is the set value that is defined by the
process characteristics. Even though VS/TS ratio went through
an up and down evolution, it fluctuated between 0.833 and
0.933 (average of 8.77). Furthermore, low amount of mixed
liquor non-volatile suspended solids (MLNVSS = 1.38 g/L in
TS = 15.2 g/L) showed no mineral accumulation owing to the
use of ultrafiltration membrane.
2.2.2. COD removal
High concentration of activated sludge resulted in achieving
high COD removal (Fig. 2). During acclimation phase,
especially in the beginning of each phase in assimilation
period, high outlet COD was reported. It seems that the high
DEHP load rate in the beginning of each phase, adsorbed by
sludge, partially interrupted the metabolism of microorgan-
isms and deactivated sludge, resulting in higher production of
SMP. Besides these peaks, the removal rate during the course
of experiments was more than 93%. According to Table 2, the
COD removal efficiency was almost independent of sludge
concentration and HRT, even though in treatment of a
complex industrial wastewater e.g., petroleum refinery, vari-
ation of HRT influences on COD removal efficiency (Reif et al.,
2008). The highest COD removal efficiency was observed
during HRT of 4 hr (outlet COD less than 1 mg/L). High organic
loading rate developed the microorganisms' community and
stimulate them to use the residual COD. Moreover, at low F/M
ratio, utilization of SMP by sludge as a food source enhanced
the COD removal efficiency. It could be also deduced that even
0.6 mg/(L·day) of DEHP load was not toxic for overall growth of
microbial community.
2.2.3. Removal of DEHP
According to Fig. 3, high DEHP removal efficiency during
continuous process by the SMBR could be distinguished.
This phenomenon can relate to the bioavailability of DEHP.
Generally, the bioavailability of DEHP is highly dependent on
the size, and degradability of material that carry it. For the real
municipal wastewater, 95.6% and 90.3% of the measured

30 1.0

25 0.9

20 0.8
HRT= HRT=8 hr HRT=4 hr
Concentration (g/L)

6 hr
VS/TS ratio

15 0.7

10 0.6
HRT= HRT=6 hr HRT=6 hr HRT=6 hr HRT=6 hr HRT=4 hr
8 hr
5 DEHP= DEHP= DEHP= DEHP= DEHP= TS 0.5
20 µg/L 20 µg/L 50 µg/L 100 µg/L 150 µg/L VS
VS/TS
0 0.4
0 20 40 60 80 100 120 140 160 180 200
Time (day)

Fig. 1 – TS, VS and TS/VS ratio evolution during experimental phases (assimilation and operating condition changes).
TS: total solid; VS: volatile solid.
42 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 37–5 0

600 70
Inlet
Outlet
60
500

50
HRT=8 hr
Inlet concertration (mg/L)
400

Outlet concertration (mg/L)

HRT=6 hr HRT=6 hr HRT=6 hr HRT=6 hr HRT=4 hr HRT=6 hr HRT=8 hr HRT=4 hr
DEHP=
20 µg/L DEHP= DEHP= DEHP= DEHP= TS=15.5 g/L TS=5.5 g/L TS=8.5 g/L TS=11.5 g/L
20 µg/L 50 µg/L 100 µg/L 100 µg/L 40
300
TS~11.5 g/L
30

200
20

100
10

0 0
0 20 40 60 80 100 120 140 160 180 200
Time (day)

Fig. 2 – Inlet, outlet COD evolution during experimental phases (assimilation and operating condition changes). COD: chemical
oxygen demand.

DEHP were associated with particles with average size of
40.7 μm in the influent and effluent, respectively (Roslev et al.,
2007). Cheng et al. (2010), estimated 68% of dissolved DEHP
(adsorbed to particle smaller than 0.4 μm) removal, in
comparison with very low efficiency for adsorbed DEHP into
suspended solid in both activated sludge (13%) and rotating
biological contactor (21.5%) processes (Cheng et al., 2010). As
ultrafiltration barricades the passage of adsorbed DEHP, high
DEHP removal efficiency could be expected by MBR. In our
synthetic wastewater, 15 mg/L of methanol was used as a
carrier of DEHP. It later biodegraded, making DEHP highly
bioavailable.
Secondly, high VS content in the aeration basin could be the
main reason for the high removal of hydrophobic compounds,
such as DEHP. Although for hydrophilic compounds the
removal is totally dependent on biodegradability, high removal
efficiency of hydrophobic compounds with logD > 3.2 (surpass
80% in biological treatment process) connects to its affinity for
adsorption (Alturki, 2013; Boonyaroj et al., 2012b; Tadkaew et al.,
2011). Huang et al. (2008) mentioned the importance of
two factors i.e., HRT and SRT in the removal of DEHP by
anaerobic–anoxic–aerobic system. Accordingly, HRT and sludge
Table 2 – Di 2-ethyl hexyl phthalate (DEHP) and COD
removal efficiency and outlet total solid (TS) and volatile
solid (VS) in different operating conditions.
HRT (hr)
4.9–6.5
DEHP removal (%) 8 77.8–92.8
6 42.7–95.0
4 –
COD removal (%) 8 94.7–97.0
6 92.7–99.0
4 –
Average TS in 6 –
effluent (mg/L) 4 –
Average VS in 6 –
effluent (mg/L) 4 –
concentration were selected as the efficacious operational
parameters for overall DEHP removal. In Table 2, the removal
efficiency ranges for these parameters illustrated independence
of HRT effect on the removal of DEHP, which was in accordance
with prior studies (Huang et al., 2008; Sakiti et al., 2013). Sludge
concentration, on the other hand, seems to be influential factor
for the removal of DEHP. In HRT = 4 hr, and VS = 14.5 ± 1.3 g/L,
the removal was mostly higher than 96% which is compatible
with the result of Sakiti et al. (2013). Except sorption into sludge,
volatilization could be a potential removal of DEHP in the
aeration basin. Eq. (1) shows the portion of volatilization in
removal of DEHP.
HCG
Air stripping removal ¼  100% ð1Þ
P atm  X  L
In which, H, is the Henry's low constant for DEHP = 4.43
(Pa·m3)/mol, G is the air flow rate of 26.8 mol/hr (2.5 L/min), C
is the hypothetical concentration of DEHP in aeration basin
(50 μg/L), L is the wastewater flow rate 55.56 mol/hr (1 L/hr),
Patm = 101,325 Pa, and X is the mole fraction of DEHP in
wastewater. If gas flow was saturated with DEHP (ideal
condition) only 0.058% of DEHP was removed which was also
calculated by O'Grady et al. (1985). It is worth mentioning that
the lab scale was run without sludge as a control experiment.
The whole DEHP removal by air stripping, adsorption to the
inlet and outlet pipe and retention with membrane was
calculated around 0.47%.
TS (g/L) All in all, under steady state condition, lower HRT increases
the sludge concentration resulting in more adsorption of DEHP
11.36–11.55 13.6–15.8
and increase in DEHP removal efficiency, even if it increases the
– – DEHP load into 0.9 μg/(L·day). It is worth mentioning that the
70.6–98.3 – removal efficiency in this study did not necessarily represent a
96.8–98.5 88.9–96.7
complete mineralization of the compound.
95.4–96.0 –
96.7–99.9 98.9–99.9
95.6–99.9 99.4–99.9 2.2.4. Effect of soluble microbial product on the removal of DEHP
817 756 EPS are sticky macromolecules produced by an active bacterial
548 442 secretion or cell lysis, which bound the sludge floc together.
118 67 SMP and extractable EPS (e-EPS) are two different kinds of
94 43 EPS made up of carbohydrates, proteins and humic substance
 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 3 7–5 0 43

200
180 Inlet

DEHP concentration (µg/L)

Outlet
160
140
HRT=6 hr
120
DEHP=150 µg/L HRT=4 hr HRT=6 hr HRT=8 hr HRT=4 hr
100
TS=11.5 g/L TS=15.5 g/L TS=5.5 g/L TS=8.5 g/L TS=11.5 g/L
80
60
40
20
0
75 95 115 135 155 175 195
Time (day)

Fig. 3 – Inlet and outlet DEHP evolution during experimental phases (assimilation and operating condition changes). DEHP: Di
2-ethyl hexyl phthalate.

(Zuriaga-Agustí et al., 2013). Diverse kinds of groups in EPS, such mineral, escaping organic matter in outlet was inevitable. In the
as aliphatic, aromatics, hydroxyl and carboxylic groups con- beginning of reactor operation with flow rate of 16 to 24 L/
tribute to the amphoteric characteristic, due to electrostatic day, detection of high concentration of DEHP in outlet even
interactions and hydrogen bonding with the hydrophilic and up to 74 μg/L was related to the sudden increase of SMP.
London force with hydrophobic compound, especially DEHP (Xu Furthermore, detected COD in the outlet was also related to
et al., 2011). It seems that polysaccharides could be used for the free SMP, as inlet glucose in the synthetic wastewater
metal removal (Tian et al., 2006), while proteins are capable of was totally biodegraded. According to Fig. 4 and Table 2, the
DEHP adsorption. According to our data, the ratio of SMP/sludge removal of COD and DEHP was related to the loading rate
fluctuated between 2.2% and 9% for VS concentration of 15.8 and SMP concentration in outlet. High removal of DEHP and
and 5.7 g/L, respectively. High large surface area of SMP (0.8– COD was observed in higher concentration of sludge (in low
1.7 m2/g) (Rogers, 1996) provides enough site for the adsorption F/M ratio), as MLSS in aeration basin consumed SMP, or
of DEHP that competes by the activated sludge, consequently entrapped them in microorganism floc (Marttinen et al.,
effect on DEHP removal (Peterson and Staples, 2003). Previous 2003).
studies on SMP in activated sludge showed that their molecular Through Table 3, it could also be inferred that in the same
weight varied in the range of 13.7 to 670 kDa (Simon et al., 2009). situation, the removal of DEHP is closely dependent on the
The range size of proteins, accordingly, differed from 3.35 to membrane pore size, because of the ability of the membrane to
12.22 nm. Therefore, the free proteins in mixed liquor could block the passage of SMP. Generally, the hollow fiber membrane
escape from membrane (pore size = 40 nm). According to with pore size less than 0.05 μm showed high DEHP removal
Table 2, the average amounts of outlet TS and VS during efficiency (more than 95%), while it was reported below 25% for
continuous mode were 648 ± 134, and 106 ± 12 mg/L, respec- the flow sheet membrane with pore size around 0.4 μm (Sakiti
tively. High VS concentration in outlet showed that except et al., 2013).

0.80 100

0.70

80
0.60
F/M ratio (kg COD/(kg VS.day))

HRT=8 hr HRT=6 hr HRT=6 hr HRT=6 hr HRT=6 hr HRT=4 hr HRT= HRT=8 hr HRT=4 hr

0.50 DEHP= DEHP= DEHP= DEHP= DEHP=150 µg/L TS=15.5 g/L 6 hr TS=8.5 g/L TS=11.5 g/L
20 µg/L 20 µg/L 50 µg/L 100 µg/L TS=5.5 g/L 60
TS=11.5 g/L
Removal (%)

0.40

40
0.30

0.20
20
F/M ratio
0.10 COD removal
DEHP removal
0.00 0
0 20 40 60 80 100 120 140 160 180 200
Time (day)

Fig. 4 – Comparison of DEHP and COD removal according to the F/M ratio in different operating conditions and different
concentration of DEHP. DEHP: Di 2-ethyl hexyl phthalate; COD: chemical oxygen demand.
44 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 37–5 0

Table 3 – DEHP removal investigation on different wastewater treatment plants.

Biological processes Initial conditions Operation Removal References
condition

Two stage (anoxic–aerobic) Landfill leachate MLSS = 11 g/L Total = 96% Boonyaroj et al.
HF-MBR (pore size: 0.4 μm), COD = 9306 mg/L HRT = 12 hr Soluble = 66% (2012a)
Thailand TKN = 218 mg/L SRT = 2500 day COD = 81%
DEHP = 65 μg/L soluble NH+4 = 83%
440 mg/kg solid TKN = 29%
(1) HF-MBR (pore size: Municipal wastewater SRT = 20 day DEHP (1) = 70% Camacho-Muñoz
0.05 μm), DEHP (average, max) = 37.2, TSS(1) = 6–9 g/L DEHP (2) = 37% et al. (2014)
(2) FS-MBR (pore size: 0.4 μm) 379 μg/L TSS(2) = 4–7 g/L
Seville, Spain HRT(1) = 12 hr
HRT(2) = 16 hr
(1) HF-MBR, Municipal wastewater MLSS = 8 g/L DEHP(1) = 57% ± 44% Cases et al. (2011)
(2) FS-MBR Alicante, Spain DEHP = 5–100 μg/L COD(1) = 91%
DEHP(2) = 56% ± 31%
COD(2) = 92%
Submerged FS-MBR Mixed industrial wastewaters HRT = 8–16 hr COD = 90.3%, ( 80%-95%) Llop et al. (2009)
(pore size: 0.4 μm) COD = 3717, SS = 250, N = 195, MLSS = 10–15 g/L Ntot-N = 78.1%
Tarragona, Spain P = 2.1 mg/L Ptot-P = 70%
DEHPin = 65.5 μg/L DEHP = 58.8%
FS-MBR (pore size = 0.1 μm), Synthetic Municipal Wastewater HRT = 24,36 hr COD (average, range) = 77%, Sakiti et al. (2013)
Thailand DEHPin range = 47–82 μg/L MLSS = 3, 6 g/L 60%–98%
DEHP (average-range) = 15%,
10%–29%
UF-MBR (100 kDa pore size), Municipal wastewater MLSS = 16 g/L MBR DEHP = 75% Melo-Guimaraes
Mexico City, Mexico DEHP = 10.3–17.5 μg/L SRT = 21 days et al. (2013)
HRT = 5 hr
WWTP based on AS, Mexico Municipal wastewater MLSS = 3 g/L COD = 89.1% Melo-Guimaraes
City, Mexico DEHP = 10.3–17.5 μg/L HRT = 80 hr N tot-N = 78.5% et al. (2013)
P tot-P = 26.5%
DEHP 46%–49%
WWTP based on AS, Paris, Municipal Wastewater HRT = 6.5 hr COD = 89% (84%–92%) Dargnat et al.
France COD (average-range) = 1000 NH+4 = 94%–97% (2009)
(777–1643) mg/L DEHP
NH+4 = 64 56–68 mg/L Primary = 52.7%
DEHP = 22.46 ± 13.22 μg/L Secondary = 25.4%
WWTP based on AS, DEHP = 98–122 μg/L SRT = 14 days DEHP = 94%, Marttinen et al. (2003)
Finland COD = 580 mg/L HRT = 20 hr 29% biodegradation
NH+4-N = 49 mg/L COD = 93%
NH+4 = 97%
WWTP by two stage AS, Municipal wastewater HRT = 1 day DEHP out = 4.92 μg/L Roslev et al. (2007)
Aalborg, Denmark DEHP = 71.89 (53.2–84.1 μg/L) SS = 4–7 g/L (2–10 μg/L)
SRT = 21–28 days 93%
81.4% biodegraded
WWTP based on AS, Municipal wastewater MLVSS = 4.2 g/L COD = 80% Cheng et al. (2010)
Hsinchu, Taiwan DEHP = 5.83–12.05 μg/L (dissolved) SRT = 20–25 days DEHP, primary clarifier 29.3%
4.55–8.81 mg/kg HRT = 2.5 hr (dissolved), 23.1% (adsorbed)
(adsorbed) DEHP, aeration basin
(dissolved) = 15%,
(adsorbed) = 32%
Industrial scale RBC, Hsinchu, DEHP = 5.83–12.05 μg/L HRT = 1.6 day DEHP (dissolved) = 50.4% Cheng et al. (2010)
Taiwan 4.55–8.81 mg/kg OLR = 1.8 g/(L.day) DEHP (adsorbed) = 58.2%
WWTP based on trickling DEHP = 23.6 ± 12.2 μg/L DEHP = 1%–44% 6.8% Oliver et al. (2005)
filter, Portsmouth, UK Max DEHP = 246 μg/L primary, 33.6% (biofilm)
COD = 745 mg/L COD = 96%
NH+4-N = 26 mg/L NH+4 = 99.2%

AS: activated sludge, FS-MBR: flow sheet-membrane bioreactor, HF-MBR: hallow fiber-membrane bioreactor, RT: hydraulic retention time,
MLSS: mixed liquor suspended solid, SRT: sludge retention time, TKN: total kjeldahl nitrogen, UF-MBR; ultrafiltration- membrane bioreactor,
WWTP: wastewater treatment plant.

2.2.5. Mass balance of DEHP in SMBR decreases from 500 to 16 and 37 μg/L for activated and
The batch experiment was performed for figuring out the deactivated sludge, respectively. Furthermore, in less than
importance of DEHP adsorption on the sludge. According to 4 hr the adsorption equilibrium was reached, which showed
Fig. 5, in the presence of 8.83 g VS/L, DEHP concentration high adsorption rate in comparison with biodegradation
 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 3 7–5 0
500
DEHP concentration (µ g/L)
450
400
350
300
250
200
150
100
50
0 dation rate of DEHP on activated sludge process with MLSS of
0 2 4 6 8 10 12 14
Time (hr)
Fig. 5 – Adsorption of 500 μg/nL of DEHP activated and
deactivated sludge (TS = 9.796 g/L, VS/TS = 0.902). DEHP: Di
2-ethyl hexyl phthalate; TS: total solid; VS: volatile solid.
rate. The removal mechanism of DEHP also was reported by
Boonyaroj et al. (2012a). Sorption on sludge consists of
adsorption to surface and partitioning into cells. For DEHP
with high octanol-water partition coefficient (Kow), partitioning
rate surpass the adsorption, as Kow effect on partitioning is
more than adsorption. Therefore, the accumulation of DEHP on
surface of sludge saturated the active sites in surface (0.82–
1.66 m2/g), especially in MBR with long SRT (Wang et al., 1993).
Despite this consideration, the rate of adsorption surpasses the
rate of biodegradation. In our MBR, in the end of day 108, the
concentration DEHP inside the sludge was around 1497.9 μg
DEHP/g VS dry weight, which surpasses the norm concentra-
tion 1.34–850 μg/g dry weight (Boonyaroj et al., 2012a; Cheng
et al., 2000; Huang et al., 2008); yet, lower than the highest
uptake (58,300 μg/g VSS) (Oliver et al., 2005). According to this
data, the mass balance of SMBR was figured out in the time
period of days 100 to 108. According to Fig. 6, just around
23.33% of inlet DEHP was discharged by the sludge. Even
though DEHP was categorized as slow degradation compound
due to its electron withdrawing groups, highly assimilated
microorganism removed around 34 μg DEHP/(g VS·day)
DEHP/(g VS.day)
107.2 µg/L
518.15 µg DEHP/(L .day)
2.89 mg DEHP/day
Fig. 6 – Mass balance of DEHP in SMBR during the days 100 to 108. DEHP: Di 2-ethyl hexyl phthalate; SMBR: submerged
membrane bioreactor.
45
(73.95%). In the study on Denmark WWTP, it was reported
Deactivated sludge
Activated sludge that high biodegradation rate decreases the concentration of
DEHP in sludge (3.51 mg/kg). From 35.4 μg/L of inlet DEHP,
71% was biodegraded and 28% was discharged in sludge
(Fauser et al., 2003). Roslev et al. (2007) also stated that the
Adsorption = 92.6% concentration of DEHP in activated sludge is in the range of
Biodegradation = 4.2% 61.3–77.9 mg/kg meaning 81.6% of biodegradation occurred
inside the sludge. Estimation of Clark et al. (1995) on biodegra-
16 18 20 22 24 2 g/L showed the biodegradation rate of 0.083 L/(g VSS·day)
(Clark et al., 1995). By considering this number, the half-life of
the bioreactor under the optimum operating condition was in
the range of 7–8 hr.
In spite of high biodegradation rate, long SRT in our case
increases the DEHP concentration in sludge 15 times than the
allowable concentration of DEHP in North American regula-
tion (100 μg/g dry weight) (Marttinen et al., 2004). According
to the three viable mechanisms of DEHP removal, Fig. 7
schematically represents the possible fate of DEHP in the
membrane bioreactor.
2.2.6. Nitrogen and phosphorous evolution in MBR
Aerobic processes with the SRT more than 25 days ensured
the growth of chemoautotrophic nitrifying bacteria within the
bioreactor independent of sludge concentration (Mohammed
et al., 2008). Hence, our MBR with SRT of 140 days provided
ideal niche for proliferation of nitrobacteria, which has a long
generation time. By closely looking at Fig. 8, the enhanced
removal efficiency of ammonium is evident (the average
concentration of 0.22 mg/L NH+4). Lower sludge production and
good aeration (DO always more than 3 mg O2/L), nitrifying
bacteria face less competition from other heterotrophic
causing effluent ammonia to drop to less than 1 mg NH+4/L.
During the entire experiment, the concentration of ammonia
rarely exceeded 0.5 mg/L. The first period occurred by introduc-
tion of 0.08 mg DEHP/(L·day), and the second one by organic
load of 0.6 mg DEHP/(L·day), and the third occurred when
Volatilization
34 µg adsorption by
membrane and
reactor wall
2.4 µg/L
SMBR 9.53 µg DEHP/(L .day)
11364 mgVS/L 0.038 g DEHP/day
Outlet by sludge
23.33%
324.7 mg VS/day
1497.9 µg DEHP/g VS dry weight
486 µg DEHP/day
46 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 37–5 0

Inlet
DEHP

Adsorbed to suspended solid

Dissolved DEHP,
or organic materials
below 3 µg/L
up to 150 µg/L

Biodegradation Adsorption Leaching out of

Inert carrier
of carrier of carrier into cell suspended solid

Non-
absorbable Absorbable

Adsorb Adsorb
Biodegradation
by EPS by sludge

Membrane
No
rejection
Yes Accumulation in Exit by
Exit by
effluent aeration basin sludge drawl

Fig. 7 – Prediction of DEHP fate in the membrane bioreactor. DEHP: Di 2-ethyl hexyl phthalate.

the concentration of microorganisms rapidly increased in the concentration of NOx, mainly NO−3 in the effluent was
reactor. According to the sensibility of nitrobacters, either inevitable. During close investigation of total nitrogen (TN)
slight shock by DEHP or competition from other autotrophic removal in SMBR, especially after assimilation period, higher
bacteria affected their performance. However, the variation portion of ammonia in untreated wastewater was directly
of effluent ammonia was not distinguishable. As predicted by removed by air stripping as NH3 gas. According to the W.P.C.F.
Tadkaew et al. (2011), one step aerated basin does not have (1983), at 20°C and pH more than 7, the part of NH+4 was
any biological denitrification capability. As a result, elevated transfered into NH3 which later removes from the aeration

Nitrogen load rate Ammonia removal TN removal

8.2 100

90
8.0
Nitrogen load rate (g N/(L.day))

80
7.8
HRT=8 hr HRT=6 hr HRT=6 hr HRT=6 hr HRT=6 hr HRT=4 hr HRT=4 hr HRT=8 hr HRT=4 hr 70
7.6 DEHP= DEHP= DEHP= DEHP= DEHP= TS=15.5 g/L TS=5.5 g/L TS=8.5 g/L TS=11.5 g/L
Removal (%)

60
20 µg/L 20 µg/L 50 µg/L 100 µg/L 150 µg/L
7.4 50
TS=11.5 g/L
40
7.2
30
7.0
20
6.8
10
6.6 0
0 20 40 60 80 100 120 140 160 180 200
Time (day)

Fig. 8 – Ammonia and total-nitrogen evolution during experimental phase (assimilation and operating condition changes).
 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 3 7–5 0
basin by air stripping. During the continuous mode, the SMR
worked in pH more than 7.5, even up to 7.9 in which the ratio
of free ammonia/ammonium was 3.4% (Eqs. (2) and (3)).
NH4 þ þ OH− →NH3 þ H2 O K ¼ 2:18  10−5 at temperature ¼ 20 C
n o−1
Free NH3
¼ 1 þ 100:09018þ½Tþ273:15−pH
2729:92
 100%
Total ammonia
Since ammonia constantly escape from the aeration basin,
Eq. (1) was always unbalance and went to the production of
more ammonia; hence, even 85% of TN average removal
efficiency was achieved. Another hypothesis for the high TN
removal efficiency could be conversion of soluble carbon and
nitrogen source into insoluble biomass (Nghiem et al., 2009).
Moreover, phosphorous accumulating organisms (Fatone et
al., 2005) are only developed in the anaerobic condition
so that they assist the next aerobic process for storage of
phosphorous in the form of poly phosphorous by production of
polyhydroxybutyrate from volatile fatty acids. The aeration basin
was obviously unable to support these bacteria; however sludge
assimilated around 6 mg P/g COD. As a result, around 2.5 mg P/L
was required for bacterial growth leading to have a phosphorous
removal in the range of 40% to 50% (3.5 ± 2 mg P/L in effluent).
As seen in Fig. 9, for the days 107 and 151, strangely the
concentration of effluent exceeded the influent, which can be
related to the sudden release of polyphosphate as well as SMP.
2.3. Fouling
In these sets of experiment, transmembrane pressure was
continuously checked as an indicator of fouling phenomena.
It was thought that the membrane flux, concentration of sludge,
organic suspended solids, sudden release of EPS from the
bacterial cells, accumulation of mineral, and mean floc size
are pivotal factors for clogging of membrane (Ying et al., 2010).
Although fouling rate seems to be decreased in high SRT and
MLSS (Van den Broeck et al., 2012), the severe membrane fouling
was observed during operation of MBR at the end of HRT = 4 hr
stage with the average concentration of 15 g VS/L. According to
Fig. 10, whenever the concentration of COD in supernatant
increased more than 110 mg/L, noticeable increase in the rate of
membrane fouling was observed. Decreasing HRT from 6 to 4 hr
10
9 HRT=6 hr
DEHP=
8 50 µg/L
7
6
Inlet (mg/L)
5
4
3
HRT=8 hr HRT=6 hr
2 DEHP=
DEHP=
20 µg/L
1 20 µg/L
0
0 10 20 30 40 50 60
Fig. 9 – Inlet and outlet PO4-P evolution during experimental phase (assimilation and operating condition changes).
47
on day 108 exacerbated the membrane clogging, decreased
dramatically the period of chemical wash. Enhanced organic
load in the range of 2.7 g COD/(L·day) rapidly increased the
sludge concentration, which required more aeration to keep OD
in the range of 2 mg/L. Slight increase in aeration broke the
ð2Þ sludge floc, released a portion of attached EPS, and consequent-
ly increased the COD of supernatant. Simultaneous increase in
ð3Þ both membrane fluxes into 21.28 L/(m2·hr) and polysaccharide
(attached EPS) concentration in supernatant of MBR (Drews
et al., 2008), triggered the fouling of membrane; consequent-
ly, the chemical wash periods decreased from every 12 days
per average to every 5 days.
2.4. Inlet and outlet toxicity
Microtox and Daphnia bioassay tests were carried to estimate
the toxic effect of synthetic wastewater samples under opti-
mum experimental conditions. Toxicity measurements record-
ed before and after treatment are summarized in Table 4. At
exposure time of 15 min, the inhibition percentage of V. fisheri
measured in the inlet solution was 50%, whereas at the outlet of
SMBR the inhibition of bioluminescence was quite nil. Besides,
toxicity results showed that the outlet effluent had no influence
on D. magma (toxicity lower than 1 TU), whereas 15 TU was
recorded for the inlet sample (untreated synthetic wastewater).
Traces of toxic metals, NH4 and DEHP are considered as a
main source of toxicity in influent. In optimum condition the
MLNVSS was measured around 1.672 g/L that showed the
adsorption of mineral, including trace metals into the sludge.
Furthermore, the complete removal of ammonia by nitrification
and air striping in SMBR, as well as adsorption and biodegrada-
tion of DEHP into the sludge removed these compounds, and
consequently no toxic effect was recorded for treated water.
3. Conclusion
The gradual growth of special degrading microorganism
community in SMBR was provided for DEHP removal. The
synthetic wastewater used in this project simulated the
composition of municipal wastewaters. Utilization of highly
biodegradable source of carbon as well as DEHP carrier facilitated
the investigation of decisive factors for the fate of DEHP in
HRT=6 hr HRT=6 hr HRT=4 hr HRT=6 hr HRT=8 hr HRT=4 hr
DEHP= DEHP=150 µg/L TS=15.5 g/L TS=5.5 g/L TS=8.5 g/L TS=11.5 g/L
100 µg/L
TS=11.5 g/L
Inlet PO4
Outlet PO4
70 80 90 100 110 120 130 140 150 160 170 180 190 200
Time (day)
48 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 37–5 0

0 180
TMP COD of supernatant HRT
HRT=8 hr 160
-10
140
HRT=6 hr
-20 120

COD (mg/L)
TMP (kPa)

100
-30 HRT=4 hr
80

-40 60

40
-50
20
-60 0
0 20 40 60 80 100 120 140 160 180 200
Time (day)

Fig. 10 – Evolution of TMP, HRT, COD of supernatant, and period of chemical wash during the time of operation. TMP: trans-
membrane pressure; HRT: hydraulic retention time; COD: chemical oxygen demand.

membrane bioreactor. The results demonstrated that under
optimum operating conditions of (HRT = 4 hr, SRT = 140 days,
temperature = 18°C, pH = 7.5, average VS = 14.29 g/L, average
F/M ratio = 0.188 g COD/(g VS·day) and DEHP load rate =
0.832 mg/(L·day)) the average removal efficiency of COD, DEHP,
NH4, PO4, and toxicity were 99.7%, 96.0%, 99.2%, 46.6%, and
100%, respectively. Although the removal efficiency of carbon
and nitrogen slightly changed under different operating condi-
tions during course of experiment, DEHP removal efficiency
distinctly depended on the sludge concentration. Among the
main operating parameters, HRT, SRT and VS, the sludge
concentration demonstrated substantial effect on the removal
of DEHP. Later on, the hypothesis of the effect of SMP on the
removal of DEHP was discussed and partially proved that their
types and sizes could affect the removal of hydrophobic
compound, especially DEHP. F/M ratio and membrane pore
were considered as the main operational factors that deter-
mined the outlet concentration of SMP. The concentration of
DEHP in sludge in the best performance of SMBR for DEHP
removal efficiency (97.75%) was around 1.5 μg/g VS dry weight,
which exceeds the discharge limit. Yet, around 74% of average
110 μg/L of inlet DEHP was biodegraded in sludge. Finally, no
toxic effect of DEHP was observed in the organic load of up to
75 μg DEHP/(g VSS·day) even in highly sensitive nitrobacteria.
The acute toxicity of synthetic wastewater and treated water
showed the significant ability of SMBR to decrease the toxic
Table 4 – Toxicity measurements before and after treatment
in optimal conditions.
Parameters Wastewater
samples
Inlet Outlet
Toxicity
Daphnia magna test (TU) 15 <1
Vibrio fischeri test (Microtox) 50% 0.0%
TU = Toxicity unit
Operating conditions: HRT = 6 hr, SRT = 140 days, VS concentration =
12.11 g/L, inlet NH+4 = 25.4 mg/L, inlet DEHP = 123.5 μg/L, inlet COD =
472.5 mg/L.
effect of wastewater and made it suitable for release to the
environment.
Acknowledgment
Sincere thanks are extended to the National Sciences and
Engineering Research Council of Canada (NSERC Grant
Number: I2IPJ 461378) for their financial contribution to this
study.
REFERENCES
Ministère de l'environnement du Québec, 2000. Détermination de
la toxicité létale CL50-48h Daphnia magna. Method MA
500-D.mag 1.0, Ministère de l'environnement du Québec,
Québec, QC, Canada.
Alturki, A., 2013. Removal of Trace Organic Contaminants by
Integrated Membrane Processes for Indirect Potable Water
Reuse Applications. School of Civil, Mining and Environmental
Engineering. University of Wollongong, p. 171.
A.P.H.A., 2005. Standard Methods for the Examination of Water
and Wastewater. 21st ed. AWWA and WEF DC, Washington.
Barnabé, S., Beauchesne, I., Cooper, D.G., Nicell, J.A., 2008.
Plasticizers and their degradation products in the process
streams of a large urban physicochemical sewage treatment
plant. Water Res. 42 (1-2), 153–162.
Boonyaroj, V., Chiemchaisri, C., Chiemchaisri, W.,
Theepharaksapan, S., Yamamoto, K., 2012a. Toxic organic
micro-pollutants removal mechanisms in long-term operated
membrane bioreactor treating municipal solid waste leachate.
Reduction of
Bioresour. Technol. 113, 174–180.
toxicity
Boonyaroj, V., Chiemchaisri, C., Chiemchaisri, W., Yamamoto,
K., 2012b. Removal of organic micro-pollutants from solid
waste landfill leachate in membrane bioreactor operated
without excess sludge discharge. Water Sci. Technol. 66 (8),
100% 1774–1780.
100% Camacho-Muñoz, D., Martín, J., Santos, J.L., Aparicio, I., Alonso, E.,
2014. Concentration evolution of pharmaceutically active
compounds in raw urban and industrial wastewater.
Chemosphere 111, 70–79.
Cases, V., Alonso, V., Argandoña, V., Rodriguez, M., Prats, D., 2011.
Endocrine disrupting compounds: a comparison of removal
 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 3 7–5 0
between conventional activated sludge and membrane
bioreactors. Desalination 272, 240–245.
Cheng, H.F., Kumar, M., Lin, J.G., 2010. Assessment of di-(2-ethylhexyl)
phthalate (DEHP) removal in a rotating biological contactor and
activated sludge process treating domestic wastewater. Sep. Sci.
Technol. 45 (2), 221–227.
Cheng, H., Chen, S., Lin, J., 2000. Biodegradation of di-(2-ethylhexyl)
phthalate in sewage sludge. Water Sci. Technol. 41 (12), 1–6.
Clara, M., Strenn, B., Gans, O., Martinez, E., Kreuzinger, N., Kroiss,
H., 2005. Removal of selected pharmaceuticals, fragrances and
endocrine disrupting compounds in a membrane bioreactor
and conventional wastewater treatment plants. Water Res. 39
(19), 4797–4807.
Clark, B., Henry, G.L.H., Mackay, D., 1995. Fugacity analysis and
model of organic chemical fate in a sewage treatment plant.
Environ. Sci. Technol. 29 (6), 1488–1494.
Dargnat, C., Teil, M.J., Chevreuil, M., Blanchard, M., 2009.
Phthalate removal throughout wastewater treatment plant:
case study of Marne Aval station (France). Sci. Total Environ.
407, 1235–1244.
Drews, A., Vocks, M., Bracklow, U., Iversen, V., Kraume, M., 2008.
Does fouling in MBRs depend on SMP? Desalination 231 (1-3),
141–149.
Environnement Canada, 1992. Essai de toxicité sur la bactérie
luminescente Photobacterium phosphoreum. Report SPE 1/RM/24
SPE 1/RM/24, Ottawa, ON, Canada.
Environnement Canada, 2000. Méthode d'essai biologique:
méthode de référence pour la détermination de la létalité
aiguë d'effluents chez Daphnia magna. Report SPE1/RM/14,
Ottawa, ON, Canada.
Fatone, F., Bolzonella, D., Battistoni, P., Cecchi, F., 2005. Removal of
nutrients and micropollutants treating low loaded wastewaters in
a membrane bioreactor operating the automatic alternate-cycles
process. Desalination 183 (1-3), 395–405.
Fauser, P., Vikelsøe, J., Sørensen, P.B., Carlsen, L., 2003. Phthalates,
nonylphenols and LAS in an alternately operated wastewater
treatment plant—fate modelling based on measured
concentrations in wastewater and sludge. Water Res. 37 (6),
1288–1295.
Fernandez, M.P., Ikonomou, M.G., Buchanan, I., 2007. An assessment
of estrogenic organic contaminants in Canadian wastewaters.
Sci. Total Environ. 373 (1), 250–269.
Huang, M.H., Li, Y.M., Gu, G.W., 2008. The effects of hydraulic
retention time and sludge retention time on the fate of
di-(2-ethylhexyl) phthalate in a laboratory-scale
anaerobic-anoxic-aerobic activated sludge system.
Bioresour. Technol. 99 (17), 8107–8111.
Llop, A., Borrull, F., Pocurull, E., 2009. Comparison of the removal
of phthalates and other organic pollutants from industrial
wastewaters in membrane bioreactor and conventional
activated sludge treatment plants. Water Sci. Technol. 60,
2425–2437.
Magdouli, S., Daghrir, R., Brar, S.K., Drogui, P., Tyagi, R.D., 2013. Di
2-ethylhexylphtalate in the aquatic and terrestrial environment:
a critical review. J. Environ. Manag. 127, 36–49.
Marttinen, S.K., Hänninen, K., Rintala, J.A., 2004. Removal of DEHP
in composting and aeration of sewage sludge. Chemosphere 54
(3), 265–272.
Marttinen, S.K., Kettunen, R.H., Sormunen, K.M., Rintala, J.A., 2003.
Removal of bis (2-ethylhexyl) phthalate at a sewage treatment
plant. Water Res. 37 (6), 1385–1393.
Melo-Guimaraes, A., Torner-Morales, F.J., Duran-Alvarez, J.C.,
Jimenez-Cisneros, B.E., 2013. Removal and fate of emerging
contaminants combining biological, flocculation and
membrane treatments. Water Sci. Technol. 67, 877–885.
Mohammed, T.A., Birima, A.H., Noor, M.J.M.M., Muyibi, S.A., Idris,
A., 2008. Evaluation of using membrane bioreactor for treating
municipal wastewater at different operating conditions.
Desalination 221 (1-3), 502–510.
49
Nghiem, L.D., Tadkaew, N., Sivakumar, M., 2009. Removal of trace
organic contaminants by submerged membrane bioreactors.
Desalination 236 (1-3), 127–134.
O'Grady, D.P., Howard, P.H., Werner, A.F., 1985. Activated sludge
biodegradation of 12 commercial phthalate esters. Appl.
Environ. Microbiol. 49 (2), 443–445.
Oliver, R., May, E., Williams, J., 2005. The occurrence and removal
of phthalates in a trickle filter STW. Water Res. 39 (18),
4436–4444.
Olujimi, O.O., Fatoki, O.S., Odendaal, J.P., Daso, A.P., 2012.
Chemical monitoring and temporal variation in levels of
endocrine disrupting chemicals (priority phenols and
phthalate esters) from selected wastewater treatment plant
and freshwater systems in Republic of South Africa.
Microchem. J. 101, 11–23.
Peterson, D.R., Staples, C.A., 2003. Degradation of phthalate esters
in the environment. In: Staples, C.A. (Ed.), Series Anthropogenic
Compounds. Springer, Berlin Heidelberg, pp. 85–124.
Reid, A.M., Brougham, C.A., Fogarty, A.M., Roche, J.J., 2009.
Accelerated solvent-based extraction and enrichment of
selected plasticisers and 4-nonylphenol, and extraction of tin
from organotin sources in sediments, sludges and leachate
soils. Anal. Chim. Acta 634 (2), 197–204.
Reif, R., Suárez, S., Omil, F., Lema, J.M., 2008. Fate of pharmaceuticals
and cosmetic ingredients during the operation of a MBR treating
sewage. Desalination 221 (1-3), 511–517.
Rogers, H.R., 1996. Sources, behaviour and fate of organic
contaminants during sewage treatment and in sewage
sludges. Sci. Total Environ. 185 (1-3), 3–26.
Roslev, P., Vorkamp, K., Aarup, J., Frederiksen, K., Nielsen, P.H.,
2007. Degradation of phthalate esters in an activated sludge
wastewater treatment plant. Water Res. 41 (5), 969–976.
Sakiti, S.A., Boontanon, S.K., Boontanon, N., 2013. Removal of
di-2-ethyl hexyl phthalates by membrane bioreactor.
J. Environ. Prot. 4 (4) (Article ID: 30749).
Seyhi, B., Drogui, P., Buelna, G., Blais, J.F., 2012. Removal of
bisphenol-A from spiked synthetic effluents using an
immersed membrane activated sludge process. Sep. Purif.
Technol. 87, 101–109.
Simon, S., Pairo, B., Villain, M., D'Abzac, P., Van Hullebusch, E.,
Lens, P., et al., 2009. Evaluation of size exclusion chromatography
(SEC) for the characterization of extracellular polymeric
substances (EPS) in anaerobic granular sludges. Bioresour.
Technol. 100 (24), 6258–6268.
Subramanian, S.B., Yan, S., Tyagi, R.D., Surampalli, R.Y., 2010.
Extracellular polymeric substances (EPS) producing bacterial
strains of municipal wastewater sludge: isolation, molecular
identification, EPS characterization and performance for
sludge settling and dewatering. Water Res. 44 (7), 2253–2266.
Sun, Y., Huang, H., Sun, Y., Wang, C., Shi, X., Hu, H., et al., 2014.
Occurrence of estrogenic endocrine disrupting chemicals
concern in sewage plant effluent. Front. Environ. Sci. Eng. 8 (1),
18–26.
Tadkaew, N., Hai, F.I., McDonald, J.A., Khan, S.J., Nghiem, L.D.,
2011. Removal of trace organics by MBR treatment: the role of
molecular properties. Water Res. 45 (8), 2439–2451.
Tan, B.L.L., Hawker, D.W., Müller, J.F., Leusch, F.D.L., Tremblay,
L.A., Chapman, H.F., 2007. Comprehensive study of endocrine
disrupting compounds using grab and passive sampling at
selected wastewater treatment plants in South East
Queensland, Australia. Environ. Int. 33 (5), 654–669.
Tian, Y., Zheng, L., Sun, D.Z., 2006. Functions and behaviors of
activated sludge extracellular polymeric substances (EPS): a
promising environmental interest. J. Environ. Sci. 18 (3),
420–427.
Trussell, R.S., Merlo, R.P., Hermanowicz, S.W., Jenkins, D., 2006.
The effect of organic loading on process performance and
membrane fouling in a submerged membrane bioreactor
treating municipal wastewater. Water Res. 40 (14), 2675–2683.
50 J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 7 (2 0 1 5) 37–5 0
USEPA, 1993. Methods for Measuring the Acute Toxicity of
Effluents and Receiving Waters to Freshwater, Marine
Organisms. EPA/600/4-90/027F, U.S. Environmental Protection
Agency, Cincinnati, Ohio.
Van den Broeck, R., Van Dierdonck, J., Nijskens, P., Dotremont, C.,
Krzeminski, P., van der Graaf, J.H.J.M., et al., 2012. The
influence of solids retention time on activated sludge
bioflocculation and membrane fouling in a membrane
bioreactor (MBR). J. Membr. Sci. 401–402, 48–55.
Viero, A.F., Sant'Anna Jr., G.L., 2008. Is hydraulic retention time an
essential parameter for MBR performance? J. Hazard. Mater.
150 (1), 185–186.
Wang, L., Govind, R., Dobbs, R.A., 1993. Sorption of toxic organic
compounds on wastewater solids: mechanism and modeling.
Environ. Sci. Technol. 27 (1), 152–158.
W.P.C.F., 1983. Nutrient Control, Manual of Practice. Publication
Number FD-7. Water Pollution Control Federation, Washington,
DC.
Xu, C., Zhang, S., Chuang, C.Y., Miller, E.J., Schwehr, K.A., Santschi,
P.H., 2011. Chemical composition and relative hydrophobicity
of microbial exopolymeric substances (EPS) isolated by anion
exchange chromatography and their actinide-binding affinities.
Mar. Chem. 126 (1-4), 27–36.
Ying, W., Yang, F., Bick, A., Oron, G., Herzberg, M., 2010.
Extracellular polymeric substances (EPS) in a hybrid growth
membrane bioreactor (HG-MBR): viscoelastic and adherence
characteristics. Environ. Sci. Technol. 44 (22), 8636–8643.
Zolfaghari, M., Drogui, P., Seyhi, B., Brar, S.K., Buelna, G., Dubé, R.,
2014. Occurrence, fate and effects of Di (2-ethylhexyl) phthalate
in wastewater treatment plants: a review. Environ. Pollut. 194,
281–293.
Zuriaga-Agustí, E., Bes-Piá, A., Mendoza-Roca, J.A., Alonso-Molina,
J.L., 2013. Influence of extraction methods on proteins and
carbohydrates analysis from MBR activated sludge flocs in
view of improving EPS determination. Sep. Purif. Technol. 112,
1–10.