Science of the Total Environment 693 (2019) 133425

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Bio-carrier and operating temperature effect on ammonia removal from

secondary wastewater effluents using moving bed biofilm
reactor (MBBR)
Amal Ashkanani a, Fares Almomani a,⁎, Majeda Khraisheh a, Rahul Bhosale a,
Muhammad Tawalbeh b, Khaled AlJaml a
a
Department of Chemical Engineering, College of Engineering, Qatar University, P.O. Box 2713, Doha, Qatar
b
Sustainable & Renewable Energy Engineering Department, College of Engineering, University of Sharjah, P.O. Box 27272, Sharjah, United Arab Emirates

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

• Ammonia a harmful constituent linked

to acute/chronic toxicities in surface
water
• Impact of biocarriers' characteristics on
ammonia removal by MMBR was evalu-
ated.
• MBBR technology is feasible choice to
remove ammonia from Wastewater.
• MBBR achieved effective removal of
micronutrients at low temperatures.
• Cold temperatures develop thick nitrify-
ing biofilm, less cells and lower activity.

a r t i c l e i n f o a b s t r a c t

Article history: This study investigates the impact of bio-carriers' surface area and shape, wastewater chemistry and operating
Received 4 February 2019 temperature on ammonia removal from real wastewater effluents using Moving bed biofilm reactors (MBBRs)
Received in revised form 15 June 2019 operated with three different AnoxKaldness bio-carriers (K3, K5, and M). The study concludes the surface area
Accepted 15 July 2019
loading rate, specific surface area, and shape of bio-carrier affect ammonia removal under real conditions.
Available online 16 July 2019
MBBR kinetics and sensitivity for temperature changes were affected by bio-carrier type. High surface area bio-
Keywords:
carriers resulted in low ammonia removal and bio-carrier clogging. Significant ammonia removals of 1.420 ±
Secondary effluents 0.06 and 1.103 ± 0.06 g − N/m2. d were achieved by K3(As = 500 m2/m3) at 35 and 20 °C, respectively. Lower
Performance removals were obtained by high surface area bio-carrier K5 (1.123 ± 0.06 and 0.920 ± 0.06 g − N/m2. d) and
Organic loading M (0.456 ± 0.05 and 0.295 ± 0.05 g − N/m2. d) at 35 and 20 °C, respectively. Theta model successfully represents
Total nitrogen ammonia removal kinetics with θ values of 1.12, 1.06 and 1.13 for bio-carrier K3, K5 and M respectively. MBBR
Biofilm growth technology is a feasible choice for treatment of real wastewater effluents containing high ammonia
Clogging concentrations.
Nitrification
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
⁎ Corresponding author at: College of Engineering, Department of Chemical
Engineering, Qatar University, P.O. Box 2713, Doha, Qatar. Conventional wastewater treatment plants (CWWTPs) represent
E-mail address: falmomani@qu.edu.qa (F. Almomani). the majority of treatment processes operated worldwide. CWWTPs

https://doi.org/10.1016/j.scitotenv.2019.07.231
0048-9697/© 2019 Elsevier B.V. All rights reserved.
2 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425
can effectively remove organic constituent from the wastewater but
they have a limited ability to nitrify ammonia (Nsenga Kumwimba
and Meng, 2019; Houtman, 2010; Al Momani, 2006; Al Momani et al.,
2004). Ammonia is one of the harmful constituents released from
WWTPs that is responsible for the occurrence of acute and chronic tox-
icities in lakes and rivers (Shi et al., 2019; Li et al., 2019; Judd et al., 2017;
Almomani and Khraisheh, 2016; Allagui et al., 2014; Allagui et al., 2012;
Vitse et al., 2005; Marsalek et al., 1997; Oswin and Salomon, 1963). The
wastewater effluent regulations in different countries require a mini-
mum of secondary level wastewater treatment or equivalent
(Suchetana et al., 2017; Almomani et al., 2014). In addition, the regula-
tions specify the effluent concentration limit for organic matters, solids,
and ammonia (Znad et al., 2018; AlMomani and Örmeci, 2016). Sharma
and Ahlert (1977), Welander et al. (1997), Zhu and Chen, 2002 and
Pelissari et al. (2017) have shown that the deficiencies in nitrogen com-
pound removal in conventional treatment processes are related to the
limited growth of nitrifying bacteria on suspended systems. Hence,
there is a need for advanced treatment technologies that can be used
for the removal of these constituents under real operational conditions.
Moving bed biofilm reactors (MBBRs) have shown significant am-
monia removal from wastewater under different working conditions
(Delatolla et al., 2010; Delatolla et al., 2009; Andreottola et al., 2002;
Rusten et al., 1995; Ødegaard et al., 1994; Rusten et al., 1994) and
wide range of temperatures 1–21 °C (Almomani et al., 2014; Zhang
et al., 2014; Welander and Mattiasson, 2003; Welander et al., 1997).
The MBBRs are cost-effective continual flow process that benefits from
a small footprint, ease of installation, control and operation and minimal
sludge production (90% lower than traditional WWT) (Young et al.,
2017a, 2017b). The installation of MBBR at the upstream of the
CWWTPs where the C/N ratio is low will reduce the effect of organic
matter on nitrifying bacteria and consequently promote higher rates
of nitrification. The MBBR systems do not require backwash and the op-
erational capacity can be increased by increasing the percentage fill of
bio-carrier (Liu et al., 2018). Moreover, the reported resistance to toxic-
ity spikes, shock loads and fluctuation in loading rate are also factors
adding to the advantageous use of MBBR systems (Torresi et al., 2019;
Alizadeh et al., 2019; Oleszkiewicz and Barnard, 2006).
Typically, the performance of MBBRs depends on wastewater quality
parameters, temperature, and pH (Bering et al., 2018; Abtahi et al.,
2018). Most of the published works ignored the effect of water matrix
on the nitrification process and focus on evaluating ammonia removal
using synthetic wastewater (Rodgers and Zhan, 2004a, 2004b). Al-
though the synthetic wastewater might not contain exact concentra-
tions of ammonia as real wastewater, the effects of micro-
constituents, organic matter and water matrix interference are ignored
which affects the reliability of the results. Hoang et al. (2014) investi-
gated ammonia removal from synthetic wastewater at 20 and 1 °C,
the reported average removal rates were 82.4 ± 2.5 and 14.8 ±
3.4 g N/m3.d, respectively. Experiments carried out with real wastewa-
ter (lagoon effluent) using the same experimental setup showed aver-
age removal rates rate of 260.0 g-N/m3·d and 110 g-N/m3·d,
confirming the discrepancy between real and synthetic wastewater
(Almomani et al., 2014). Temperature and ammonia loading are other
important factors to consider during the operation of the MMBRs.
Houda et al. (2015) confirmed that the nitrification treatment of efflu-
ent of urban wastewater treatment depends on ammonia loading and
temperature with higher ammonia removal observed at low ammonia
concentration (20.52 ± 4.89 mg/L) and high temperature (35.45 ±
3.18 °C). Shore et al. (2012) observed very low ammonia removal
from industrial wastewater at a temperature higher than 40 °C.
Delatolla et al. (2010) noticed a 73% reduction (0.60 to 0.16 kg-N/m3.
d) in ammonia removal as the temperature decreased from 14 to 3 °C.
The performance of MBBR under different operational conditions of
real wastewater still not well-studied. Few works have considered the
effect of organic matters on the nitrification process by MBBRs (Young
et al., 2017a, 2017b; Young et al., 2016; Almomani et al., 2014).
Furthermore, a fundamental gap in knowledge exists with respect to
the response of attached biofilm and embedded microbial communities
to effluent wastewater from real treatment processes. In Addition, the
effect of bio-carriers loading rate, shape and surface area on the system
performance is still lacking and not fully understood. To the best of our
knowledge, the effect of bio-carrier type and shape on the performance
of MBBR was investigated from organic matter perspectives (Ødegaard
et al., 1994), or using synthetic wastewater in case of ammonia removal
(Forrest et al., 2016). There are no published works that investigate the
effect of these parameters on nitrifying bacteria under real conditions.
Accordingly, this study focuses on advancing the fundamental under-
standing of the effect of different operational conditions on the ammo-
nia removal from real wastewaters. Key parameters such as bio-carrier
loading rate, size, shape and water chemistry were tested to help the
understanding ammonia removal mechanism under such realistic con-
ditions. To achieve this purpose, three lab-scale MBBRs were designed
and operated using effluents of secondary wastewater process
(ESWW). The impact of bio-carrier shapes and surface area on ammonia
removals were considered. Additionally, the study presents new knowl-
edge regarding the effects of temperature on the biofilm attachment to
the MBBR bio-carrier. The results will help in the development of funda-
mental knowledge necessary to effectively design a low-cost solution to
upgrade current DWWTPs.
2. Material and methods
2.1. Experimental setup
Ammonia removal experiments were carried out in three identical
jacketed MBBRs designated as R1, R2, and R3 as presented in Fig. 1. The
total operating volume of each reactor is 2.0 L. Each MBBR has a feed
pump, pH meter, dissolved oxygen electrode (DO), aeration pipe, dis-
charge lines, and air supply. The bio-carriers occupy 30% of the reactor
volume. The Reactor temperature was controlled by adjusting the
cooling/heating water flow rate through the jacket.
Three different types of bio-carriers (AnoxKaldness-K3,
AnoxKaldness-K5, and AnoxKaldness-M) were used in each reactor.
The specific surface area available for K3, K5 and M bio-carriers is 500
m2/m3, 800 m2/m3 and 1200 m2/m3, respectively. The feed to all the
three reactors was effluents of secondary wastewater (ESWW) of mu-
nicipal wastewater treatment plant from Doha, Qatar. The characteris-
tics of the ESWW fed to the MBBRs are provided in Table 1.
2.2. Experimental procedure
Initially, the reactors were fed with SWW and operated at 20 °C for
50 days. The SWW was prepared as per the procedure presented by
Almomani et al. (2014). The reactor temperature was increased to 30
°C and then to 35 °C keeping the feed as SWW. The feed composition
was then changed by mixing SWW and ESWW in different portion
starting with 80:20% SWW:ESWW, and increased progressively until
reached 100% ESWW. All through the startup period, the temperature
was fixed at 35 °C, hydraulic retention time (HRT) was maintained at
5.25 h and the dissolved oxygen (DO) was kept in the range
7.4–8.8 mg/L. Knowles et al. (1965) suggested keeping DO in the
MBRR as high as possible to reduce oxygen demand stress by nitrifying
bacteria. Once the feed to the MBBRs reached 100% ESWW the reactors
were operated for 160 days at different temperatures. The operation of
the MBBRs can be identified in four phases related to the operational
temperature; (1) phase 1 operated from day 0 to 40 at 35 °C,
(2) phase 2 operated from day 40 to 80 at 20 °C, (3) phase 3 operated
from day 80 to 120 at 10 °C, and (4) Phase 4 operated from day 120 to
160 at 4 °C. The operational parameters (DO, pH, average nitrogen
source (S0N), average carbon source (S0c), flow rate (Q) and hydraulic
retention time (HRT) and other parameters of the MBBRs are summa-
rized in Table 2. Samples were withdrawn daily in each period for
 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425 3

Fig. 1. Schematic of experimental set up for continues flow MBBR.

analysis. The HRTs of the reactors were controlled by adjusting the at 105 °C for 5 h, and ignited it at 550 °C for 15 min. The difference in
speed of the feed pump. Table 2 shows that the DO for all the MBBRs weight was reported as TAS and VAS, respectively. The statistical signif-
ranged from 7.4 to 8.8 mg/L, the pH variation is very small in the icance of the measured TAS and VAS with respect to average value was
range 7.7 to 8.2, average nitrogen source range from 65.2 ± 4.1 to evaluated using the Student's paired t-test. The test was conducted to
67.5 ± 4.2 gN/m3 and average carbon source range from 73.2 ± 5.5 to investigate whether the differences between the individual and average
83.1 ± 8.1 g CODdiss/m3. The feed flow rate (Q) and HRT were fixed at measurements is significant. The analysis was performed by one-way
6.40 L/day and 5.25 h. analysis of variance (ANOVA) using Prism GraphPad statistics software
(Version 7.04, USA). All statistical tests were carried out at a significance
2.3. Analytical methods level of 5%. DO was measured using Orion Star™ DO-meter (Model-
A223).
Samples withdrawn from the MBBRs on daily base were tested for The biofilm Live/Dead test was carried out using LIVE/DEAD kit
− −
ammonia (NH+ 4 − N), pH, nitrite (NO2 ), nitrate NO3 , total phosphorus (FilmTracer™) and following the procedure explained in our previous
and dissolved chemical oxygen demand (CODdiss). The total attached work (Almomani and Khraisheh, 2016). Biofilm morphology (surface
solid (TAS) and volatile attached solids (VAS) to bio-carrier were mea- coverage and thickness) was determined using a variable pressure elec-
sured on days 20, 40, 60, 80, 100, 140 and 160 of operation. The chem- tron scanning microscope (VPSEM). Measurements were performed on
ical oxygen demand (COD) was measured using HACH COD reagents 20, 40, 80, and 160 days of operation.
following the procedure outlined in the Standard Methods, test #
5220D (APHA, 1985; Eaton et al., 2005). Ammonia was tested according 3. Results and discussion
to the Standard Methods, method # 4500–NH3 B and C using Nessler re-
agent and HACH spectrophotometer (DR 2000 HACH, CO, USA) at 3.1. Feed properties
425 nm. Total phosphorous (Ptot) was measured following test #
4500-P from the standard methods (APHA, 1985). NO− 2 was tested at Fig. 2a, b and c show the total nitrogen (TN), ammonia (NH+ 4 − N),
505 nm using HACH diazotization powder pillows (Nitrite Method nitrite (NO− −
2 ), nitrate (NO3 ), COD, Ptot of the influent to MMBRs (R1, R2
8507) and HACH spectrophotometer. NO− 3 was measured at 500 nm and R3) as well as the operating temperature during 160 days of exper-
using a HACH cadmium reduction method (Nitrate Method 10020) imental phases. The water quality parameters fluctuate within the nor-
and HACH spectrophotometer. mal range as results of the change in secondary WWTP performance.
Total attached solids (TAS) and volatile attached solid (VAS) were The concentrations of TN and NH4 in the MBBRs feed ranged from
determined by scrubbing the biofilm attached to one bio-carrier, dry it 63.8 to 68.4 and 59.9 to 65 mg-N/L, respectively. The concentrations of
these two parameters although fluctuate up and down are remained
relatively constant through the operational period apart from high con-
Table 1
Water quality parameters of the effluents of secondary wastewater (ESWW) fed to the
centration value on days 40 and 60 of operation. The change in the con-
MBBRs. centrations of the feed is normal practice for treatment process dealing
with real wastewater. In addition, the fluctuating in influent water qual-
Parameter Lower value Higher value Average
ity parameters to the reactors gives a chance to test the reactor perfor-
NH3 (mgN/L) 58. 65.78 62.00 mance under varied feed concentration. Nitrate (NO− 3 ) and nitrite
NO2 (mgN/L) 0.010 0.098 0.042
(NO− 2 ) ranged from 2.99 to 3.23 and 0.0105 to 0.071 mg-N/L, respec-
NO3 (mgN/L) 2.80 3.84 3.305
COD (mgN/L) 63.27 71.04 66.96 tively; with high variation in the concentration of NO− 3 in the reactors
PO4 (mgN/L) 11.72 12.32 12.40 feed. The COD and Ptot concentrations were in the range at 62.5 to
pH 7.23 8.75 7.76 72.5 mg COD/L and 11.1 to 13.2 mg P/L. COD showed high fluctuation
C:N ratio (gCODs/gN) 1.02 1.04 1.02 during days 20 to 70 of operation. The SALR which depends on the
C:N:P ratio (gCOD:gN:gP) 4.4:5.3 4.4:5.3 4.4:5.3
bio-carries surface area is considered high for R1, medium for R2 and
4 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425

Table 2
Operational parameters of the MBBRs.

Reactor R1 R2 R3

Parameter T = 35 °C T = 20 °C T = 10 °C T = 4 °C T = 35 T = 20 °C T = 10 °C T = 4 °C T = 35 °C T = 20 T = 10 T = 4 °C
°C °C °C

Time of operation (day) 40 40 40 40 40 40 40 40 40 40 40 40

DO (mg/L) 7.4 8.3 8.6 8.8 7.4 8.3 8.6 8.8 7.4 8.3 8.6 8.8
pH 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2 7.7–8.2
Average nitrogen source (S0N,avg) 67.5 ± 67.5 ± 67.7 ± 65.2 ± 67.5 ± 67.5 ± 67.7 ± 65.2 ± 67.5 ± 67.5 ± 67.7 ± 65.2 ±
(gN/m3) 4.5 4.5 5.1 4.2 4.5 4.5 5.1 4.2 4.5 4.5 5.1 4.2
Average carbon source (S0C) (g 73.2 ± 73.2 ± 79.9 ± 83.1 ± 73.2 ± 73.2 ± 79.9 ± 83.1 ± 73.2 ± 73.2 ± 79.9 ± 83.1 ±
CODdiss/m3) 5.5 5.5 8.1 8.1 5.5 5.5 8.1 8.1 5.5 5.5 8.1 8.1
Surface area loading rate (SALR) 0.612 ± 0.611 ± 0.604 ± 0.60 ± 0.60 ± 0.382 ± 0.372 ± 0.368 ± 0.368 ± 0.25 ± 0.25 ± 0.252 ±
(g/m2.d) 0.05 0.05 0.07 0.01 0.01 0.05 0.05 0.05 0.05 0.03 0.03 0.03
Nitrogen loading rate (NLR) 305 ± 10 305 ± 10 303 ± 15 300 ± 6 300 ± 6 305 ± 10 305 ± 10 299 ± 12 299 ± 12 307 ± 309 ± 303 ± 12
(kgN/m3.day) 10 10
Organic loading rate (OLR) 313 ± 8 313 ± 8 306 ± 10 302 ± 6 302 ± 6 313 ± 8 311 ± 10 303 ± 12 303 ± 12 311 ± 8 308 ± 307 ± 12
(gCOD/m3.day) 10
Total phosphorous (g/m3) 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ± 13.1 ±
2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6
Q (L/Day) 6.40 6.40 6.40 6.40 6.40 6.40 6.40 6.40 6.40 6.40 6.40 6.40
HRT (hr) 5.25 5.25 5.25 5.25 5.25 5.25 5.25 5.25 5.25 5.25 5.25 5.25
C:N ratio (gCODs/gN) 1.2:1 1.2:1 1.3:1 1.4:1 1.2:1 1.2:1 1.3:1 1.4:1 1.2:1 1.2:1 1.3:1 1.4:1
C:N:P ratio (gCOD:gN:gP) 6.1:5.6:1 6.1:5.6:1 5.0:4.2:1 6.4:5.1:1 6.1:5.6:1 6.1:5.6:1 5.0:4.2:1 6.4:5.1:1 6.1:5.6:1 6.1:5.6:1 5.0:4.2:1 6.4:5.1:1

low for R3. Table 2 shows that SALR for R1 ranged from 0.600 to 0.612
gN/m2.day, 0.368 to 0.382 gN/m2.day for R2 and 0.250 to 0.252 gN/m2.
day for R3. The OLR and NLR for all reactors are in the same order of
magnitude ranging from 302 to 313 gCOD/m3.day and 229 to 309
kgN/m3.day, respectively. The C:N ratio ranged from 1.2:1 to 1.4:1
while the C:N:P ranged from 5.0:4.2:1 to 6.4:5.0:1.
3.2. Effect of bio-carrier on ammonia removal
Fig. 3 shows the surface area ammonia removal rate (SARR, g
− N/m2. d) as a function of time for the three bio-carriers at different
operational temperature. The SARR values were calculated at steady
state conditions using Eq. (1):
 
ðm3   9 mg/L and pH ~ 8.0 resulted in high ammonia removal (Wijffels et al.,
Q CNHþ ;in −CNHþ ;out  g 
Day 4 4
that the pH of the wastewater determine the nitrification efficiency and
suggested high ammonia removals for the wastewater with carbon to
nitrogen ratio (C/N) ratio around 1. Almomani and Khraisheh (2016)
showed that a C/N ratio in the range of 1.12 to 2.24 kgCODdiss/m3.day re-
sulted in significant ammonia removals. Higher C/N ratio might en-
hance the growth of heterotrophic bacteria and hinder the rate of
nitrification. On other hand, wastewater with low C/N ratio will have
limited heterotrophic population and high nitrification. Lazarova et al.
(1999) also suggested a C/N ratio around one for the highest ammonia
removal rate.
The average SARR for K3, K5, and M at 35 °C were 1.420 ± 0.06, 1.123
± 0.06 and 0.456 ± 0.05 g − N/m2. d. and at 20 °C were 1.103 ± 0.06,
0.920 ± 0.06 and 0.295 ± 0.05 g − N/m2. d, respectively. Previous
works showed that MBBRs operated at temperatures ≥20 °C with DO ~
1995b; Figueroa and Silverstein, 1992; Knowles et al., 1965). In the

SARR ¼   m3
2 and the DO was kept around maximum solubility limit in the range
Vr ðm Þ  As
3  %F
m3
where CNH+4 , in is ammonia concentration in the influent to MBBR, CNH+4 ,
3
outis effluent ammonia concentration from the MBBR (g-N/m ), Q is the
flow rate of feed (m3/day);Vr is the total volume of the reactor (m3),
“As” bio-carrier specific surface area (m2/m3) and %F is the percent fill
of the reactor with bio-carrier (30% in this study). The three bio-
carriers have demonstrated stable SARRs within the same temperature.
The SARRs stabilized during the first 4 days of temperature change
confirming the ability of the MBBR to respond to irregular changes in
operational conditions. Under all studied conditions, the SARR values
are different for each bio-carriers with the general trends showing
that the SARR for K3 N K5 N M. Previous studies showed a longer stabili-
zation times for nitrifiers especially at low temperatures (Andersson
et al., 2001; Wijffels et al., 1995a). Delatolla et al. (2009) and Hoang
et al. (2014) studied ammonia removals from synthetic wastewater at
4 and 1 °C and showed that the system required one to two months to
reach stable removal rate. Almomani et al. (2014) showed that ammo-
nia removal rates stabilized in the first 10 days after the temperature
change. The obtained results suggest differences in the mechanism of
ammonia removal between synthetic and real wastewater with higher
removal rate and short adaptation time observed in the case of real
wastewater. The significant ammonia removal obtained for real waste-
water can be related to the effect of micro-nutrient, organic matter and
buffering capacity of this wastewater. Whang et al. (2009) have shown
ð1Þ present study, oxygen was supplied to the MBBR via ceramic diffuser
7.5 to 8.8 mg/L and the pH was in the range 7.7 to 8.2 (Table 2), suggest-
ing that oxygen is not the limiting factor since it was at the maximum
solubility limit the conditions that enhance ammonia oxidation. The
MBBRs operated with bio-carrier K3 and K5 at T = 35 °C resulted in a sig-
nificant ammonia reduction from ~62.0 mgN/L to 8.2 and 13.4 mg-N/L,
respectively. However, the MBBR operated with bio-carrier M at the
same temperature showed an effluent ammonia concentration of
36 mg-N/L suggesting low ammonia removal. The obtained results con-
firm that the type of bio-carrier has a direct effect on ammonia removals
from wastewater. It was expected that increasing the bio-carrier specific
surface area (As) will improve the biofilm availability and thus increase
ammonia removal. However, contradictory results were observed ex-
perimentally. The MBBRs operated with bio-carrier K3 with As of 500
m2/m3 resulted in higher SARR compared with bio-carrier K5 with As
of 800 m2/m3 and M with As of 1200 m2/m3. The obtained trends
were related to low mass transfer of oxygen from water to biofilm (i.e
diffusion of oxygen) with respect to biofilm density. Bio-carrier with
high surface area showed a high biofilm density that required more ox-
ygen for ammonia oxidation. Although DO was kept at maximum in the
reactor, the mass transfer of O2 from gas to liquid for high-density bio-
film was low due to a partial or total clogging of bio-carrier cells and
thus ammonia oxidation decrease. Low surface area bio-carrier showed
low-density biofilm, high mass transfer of O2 from gas to liquid and sig-
nificant ammonia removals. Further work is required to confirm these
observations. Forrest et al. (2016) reported the same clogging problem
 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425
− −
Fig. 2. TN, NH+ 4 − N, NO2 , NO3 , CODs and Ptot of the influent to MMBRs (R1, R2 and R3)
during 160 days of experiment phases. Conditions: phase I, T = 35 °C, phase II, T = 20
°C, phase III, T = 10 °C and phase IV, T = 4 °C.
in the study carried out with the same bio-carrier using synthetic waste-
water. It was expected the NLR and OLR are the main reason for the
clogging problem. Thus, another set of experiments was conducted
with NLR of 163(kg-N/m3.day) and OLR of 150 gCOD/m3.day. Reducing
the NLR and OLR did not resolve the clogging problem but it was de-
layed; while the clogging was observed after 45 days for the bio-
carrier operated with high OLR and NLR, it was delayed till day 90 of op-
eration for wastewater with low loading. Still, bio-carrier with low As
showed higher ammonia removal rate.
Complimentary to Fig. 3, Table 3 includes the SARR values for each
reactor at each operational temperature. Table 3 also shows surface
area carbon removal rate (SACR) (kgCOD/m2.d), the average concentra-
− −
tions of NH+4 -N, NO2 -N, CODdiss, Ptot, and NO3 -N in the effluent under
steady state conditions. The average SACR was also observed to be a
function of bio-carrier surface area and operating temperature with
the highest values observed in R1 (K3) and at 35 °C. The nitrogen
mass balance (ammonia, nitrite, and nitrate) calculated during experi-
mental phases confirmed that nitrogen content in the reactors was
5
Fig. 3. Surface area ammonia removal rate (SARR, g − N/m2. d) and Arrhenius-type Theta
model regression as function of time for the three bio-carriers at different operational
temperature.
conserved and the removal of ammonia was achieved by biological con-
version. The maximum losses in total nitrogen did not exceed 4–8% for
all MBBRs.
The effluent concentrations of nitrite ranged between 0.25 and
0.35 mg-N/L, 0.22–0.86 mg-N/L and 0.25 to 0.90 mg-N/L for R1, R2,
and R3, respectively, indicating that MBBRs are capable of nitrifying am-
monia in ESWW and maintained full nitrification to NO− 3 -N at each
temperature.
The percent ammonia removal from the three reactors was also cal-
culated and showed trends similar to the SARR. The reactor operated
with bio-carrier K3 has higher % NH+ 4 − N removal compared with reac-
tors operated with K5 and M. The percentage ammonia removals at 20
°C were 87.3%, 71.8% and 47.2% for the reactors operated with K3, K5
and M, respectively. The values decreased to 59.3%, 44.6% and 12.5%
when the temperature reduced to 4 °C. The obtained percentage ammo-
nia removals are considered lower than values reported by Forrest et al.
(2016) using same bio-carriers. The differences in % ammonia removal
6 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425

Table 3
Summary of ammonia, organic and phosphorous removal rates for the MBBRs during experimental phases I-IV.

Reactor R1 R2 R3

T = 35 T = 20 T = 10 T = 4 °C T = 35 T = 20 T = 10 T = 4C T = 35 T = 20 T = 10 T = 4 °C
°C °C °C °C °C °C °C °C °C

Surface area nitrogen removal rate 1.420 ± 1.103 ± 0.95 ± 0.6424 ± 1.123 ± 0.920 ± 0.653 ± 0.423 ± 0.456 ± 0.295 ± 0.142 ± 0.05 ±
(SARR) (gN/m2.d) 0.06 0.06 0.04 0.06 0.06 0.06 0.08 0.08 0.05 0.05 0.02 0.01
RNH4 (gN/m3.d) 0.509 ± 0.494 ± 0.468 ± 0.34 ± 0.39 ± 0.408 ± 0.385 ± 0.245 ± 0.13 ± 0.10 ± 0.04 ±
0.07 0.07 0.3 0.06 0.06 0.2 0.2 0.6 0.04 0.04 0.01
Surface area carbon removal rate 0.275 ± 0.253 ± 0.1660 0.24 ± 0.130 ± 0.115 ± 0.092 ± 0.062 ± 0.074 0.062 ± 0.082 ± 0.04 ±
(SACR) (kgCOD/m2.d) 0.1 0.1 ± 0.2 0.10 0.5 0.5 0.1 0.1 0.04 0.04 0.04
Effluent NH4 (mgN/L) 8.2 ± 2.1 8.2 ± 2.1 9.4 ± 2.1 25.5.7 ± 13.4 ± 17.7 ± 19.9 ± 33.7 ± 36.0 ± 34 ± 2.1 39.3 ± 51.9 ±
5.1 1.5 2.1 4.1 5.1 2.5 3.1 4.3
Effluent NO2 (mgN/L) 0.25 ± 0.25 ± 0.35 ± 0.46 ± 0.22 ± 0.25 ± 0.55 ± 0.86 ± 0.25 ± 0.56 ± 0.87 ± 0.90 ±
0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.15 0.05 0.15 0.15 0.15
Effluent NO3 (mgN/L) 60 ± 6.0 60 ± 6.0 56.0 ± 6 42 ± 5.0 43.0 ± 48.0 ± 46.0 ± 36.0 ± 27.0 ± 32.0 ± 28.0 ± 18.0 ±
3.3 6.3 10.0 8.0 8.0 8.0 8.0 6.0
Effluent COD (mg/L) 41 ± 4 41 ± 3 48 ± 4 42 ± 3 42 ± 5 46 ± 4 52 ± 4 55 ± 5 55 ± 5 29 ± 6 31 ± 3 25 ± 4

are likely related to the effect of the feed quality parameters on re-
movals; while the present study deal with a real ESWW with a lot of
constituents that might interfere with nitrogen removal bacteria, previ-
ous studies worked with synthetic wastewater which consider the ideal
case for the ammonia removing bacteria. Luostarinen et al. (2006) re-
ported a similar percentage of ammonia removal (~60%) from real
wastewaters with nitrogen loading rate in the range of 0.012 to
0.016 kg.N/m3.d using sequencing batch mode. Whang et al. (2009)
studied the nitrification of ammonia in a high strength petrochemical
wastewater (COD = 310 mg/L and ammonia 325 mg-N/L) with C/N
ratio around 1. The average ammonia removal in the pilot-scale MBBR
was in the range 24–51%. The study suggests that reagent used in con-
trolling the pH determine the nitrification efficiency. Switching the PH
control reagent from NaOH to Na2CO3 resulted in an increase in ammo-
nia removal from 24 to 51%.
Ammonia oxidation by-products (nitrate and nitrite) were followed
on a daily base (see Table 3). The concentrations of nitrite were low in
R1& R2 ranging from 0.26 to 0.46 mg-N/L. However, in R2 the NO− 2 -N
concentration under all operational conditions was high ranging from
0.56 to 0.9. The high concentration of nitrite in the reactor may indicate
slow oxidation of ammonia and can inhibit nitrifying bacteria (Abzazou
et al., 2016; Anthonisen et al., 1976). The concentrations of NO− 2 -N in
−
the reactors is related to the oxidation of NH+ 4 − N to NO2 -N, and
then to NO− −
3 -N. The observed low concentration of NO2 -N in R1 & R2
suggests a transfer of all oxidized ammonia to nitrate–nitrogen. On an-
other hand, R3 with high nitrite concentration implies low oxidation
and low ammonia removal. The low nitrite concentrations in R1 reac-
tors were achieved by maintaining the DO in the reactor N7.5 mg O2/L
to guarantee complete oxidation of ammonia as recommended by pre-
vious works (Persson et al., 2002; Hao et al., 2002). The high concentra-
tion of NO− 2 -N inside the R2 & R3 can be related to low O2 mass transfer
rate because of bio-carrier clogging as discussed before.
NO− 3 -N concentration, on other hands, increased during the course
of experiments as results of ammonia oxidation. The concentration of
NO− 3 -N ranged from 42 ± 5.0 to 60 ± 6.0 mgN/L for R1, 48.0 ± 6.3 to
36.0 ± 8.0 for R2 and 18.0 ± 6.0 to 32.0 ± 8 mgN/L for R3. The concen-
tration of NO− 3 -N in the reactor were related to ammonia removals with
general trends indicating high NO− 3 -N for the reactor with high ammo-
nia removal and vice versa. It was also observed that decreasing the re-
actor temperature showed lower concentrations of NO− 3 -N in effluents
reflecting low ammonia removal kinetics.
The Surface Area Carbon Removal Rate (SACR) at 35 °C were 0.275,
0.130 and 0.062 kgCOD/m2.d in R1, R2 and R3. It is seen that R1 operated
with K3 with As of 500m2/m3 has the highest SACR compared with R2
and R3 operated with bio-carriers K5 and M, respectively. The SACR de-
creased by 3%, 11.5%. 16.2% when operating temperature decreased
from 35 to 20 °C. Reducing the temperature from 35 to 4 °C decreased
the SACR by 12.7%, 52.3% and 45.9% for R1, R2, and R3, respectively.
3.3. Effect of operational temperature on ammonia removal
The effect of changes in reactor temperature on ammonia removal
was highly observed in the three MBBRs. There was a significant de-
crease in SARR values when the temperature decrease from 35 to 4 °C
for all MBBRs. The SARR decreased by 54.7% (from 1.42 ± 0.06 to
0.642 ± 0.06 gN/m2.d), 62.3% (1.123 ± 0.06 to 0.423 ± 0.08 gN/m2.d
and 89.0% (0.456 ± 0.05 to 0.053 ± 0.01) for the reactors R1, R2 and
R3, respectively as the temperature reduced from 20 to 4C°. The de-
crease in ammonia removal at lower temperatures can be related to
the decrease in the relative abundance of ammonia-oxidizing bacterium
(Nitrosospira) as observed by Hoang et al. (2014). The obtained SARR as
a function of temperature agree with values in literature; Almomani and
Khraisheh (2016) reported a decrease of 40% and 24% in SARR for
MBBRs used to treat septic tank effluents at high and low HRTs (5.7
and 13.3 h). The study reports average ammonia removals of
0.540 kg-N/m3 and 0.279 kg-N/m3 using high and low HRTs (5.7 and
13.3 h) at 25 °C. Ammonia removal rates at low temperatures 4–7 °C
were in the range 0.15–0.28 kgN/m3.day as reported by Delatolla et al.
(2010). Forrest et al. (2016) studied ammonia removal from synthetic
wastewater at 25 °C, the study shows SARR values for MBBRs operated
with bio carrier K3 and M, under high loading conditions, in the range
1.85 to 1.86 kg-N/m2 and 1.56 to 1.71 kg-N/m2, respectively. Normal
loading conditions showed lower SARR in the range of 0.02 to 0.92 kg-
N/m2 and 1.56 to 1.71 kg-N/m2, respectively. Houda et al. (2015) used
fluidize nitrifying process to remove ammonia from the effluent of the
urban wastewater treatment process. The reported ammonia removal,
during 170 days of operation, was in the range 90 to 94.4%. The study
confirmed the nitrification process depends on NH4 + -N loading and
temperature with higher ammonia removal observed at low ammonia
concentration (20.52 ± 4.89 mg/L) and high temperature (35.45 ±
3.18 °C). The study showed a drop in the removal rate from approxi-
mately 0.60 kg-N/m3.d at 14 °C to approximately 0.16 kg-N/m3.d after
40 days of operation at 3 °C. Shore et al. (2012) studied ammonia re-
moval from high-temperature industrial wastewater using bench scale
MBRR operated at 35 and 45 °C. MBBRs were able to remove up to
90% of influent ammonia from industrial wastewater at temperature
range 35 and 40 °C. However, no bio-treatment was observed above
45 °C. Other studies confirmed the MBBR inability to effectively nitrify
at 45 °C (Grunditz and Dalhammar, 2001; Jones and Hood, 1980).
The kinetics of ammonia oxidation was fitted to Arrhenius-type
temperature Theta model (ATH-Model) using Eq. (2) in combination
with Eq. (3) as published by Almomani et al. (2014)
RNH4;T2 ¼ RNH4;1 :θðT 2 −T 1 Þ ð2Þ
θ ¼ 3:83x10−2 ln ðt Þ þ 9:83x10−1 ð3Þ
 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425 7

where RNH4,T1 and RNH4,T2 are the removal rates at T1 and T2 (°C),
respectively.
Ammonia removal rates for R1, R2 and R3 fitted to the ATH-Model
are presented in Fig. 3. ATH-Model fits very well the MBBR kinetics
with a coefficient of determinations (R2) N0.90 for all MBBRs. The kinet-
ics of the MBBR operated with bio-carrier K3 fits the ATH-Model with
two θ values (1.13 & 1.05), the first values applied for the temperature
range 35 to 10 °C, while the second one applies for the temperature
range 10 to 4 °C. The kinetics of ammonia removal rates for the MBBR
operated with bio-carrier K5 was fitted to ATH with three θ values
1.08, 1.03 and 1.01 for the temperature ranges 35 °C to 20 °C, 20 °C to
10 °C, and 10 °C to 4 °C respectively. The MBBR operated with bio-
carrier M fits the ATH with two values of θ of 1.02 and 0.99 for the tem-
perature ranges 35 °C to 20 °C and 20 °C to 4 °C, respectively The re-
ported θ values agree with values reported by Almomani and
Khraisheh (2016), Almomani et al. (2014), Dellatola et al. (2012), and
Salvetti et al. (2006). The variation in the value of θ for the same
MBBR under different temperatures provides information regarding
the reactor sensitivity to change in operational temperature. It is
known that MBBR with one θ value suggests that the reactor is stable
under all operating temperatures. However, change in θ values as the
reactor temperature change implies a change in ammonia removal ki-
netic as a function of temperature. The results of this study showed
two different values of θ for MBBRs operated with bio-carriers K3 and
M. The range of temperature where each kinetics dominant can be
used to determine the best operating condition for each bio-carrier.
While R1 is stable in the range 35 °C to 10 °C, reactor R3 is stable for
the temperature range 20 °C to 4 °C suggesting that the first reactor
can be operated at high temperature and the second reactor at low tem-
perature. It was also concluded that R2 is very sensitive to the tempera-
ture and it is not recommended to be used if a frequent change in
operational temperature is expected.
Fig. 4. (a) TAS and VAS of biofilm and (b) the BVRR on day 30, 60 and 150 of operation for
the MBBRs (R1, R2 and R3).
3.4. Characteristics of attached biofilm

Fig. 4a illustrates TS and VAS of the biofilm attached to the bio-
carriers on day 0, 5, 20, 40, 60, 80, 100, 140 and 160 of operation. The
time of analysis was selected to cover different operating temperatures
(35 °C, 20 °C, 10 °C and 4 °C). There was an increased in TAS and VAS of
the biofilm for all the MBBRs after 5 days following the change from
SWW to ESWW. After that, the values slightly decrease to stabilize
around an average value. The small increase and fast re-stabilization
in biofilm (TAS and VAS) after the change in feed confirms the ability
of MBBRs to respond to system upsets and/or irregular changes in
feed conditions without a major change in biofilm characteristics. In ad-
dition, it was confirmed that the biofilm was stable over the 160 days of
operation even with changes in temperature. The percentage loss from
the biofilm was very low and did not exceed 4–7% under all conditions.
The high values of TAS and VAS during the first 5 days of operation is
mainly due to change in the feed characteristics, not the temperature.
The average values of TAS (standard deviation) as well as the VAS/TAS
ratio were 70.2 mg/carrier (0.007) and 0.73(0.02) in R1, 77.4(0.03)
mg/carrier and 0.75.6(0.002) in R2 and 60.2 mg/carrier (0.005) and
0.74(0.02) in R3. The Student's paired t-test at a significance level of
5% showed that biofilm thickness measured from different bio-carrier
is not significantly different from the reported average value. These
values are slightly different at different operational temperatures.
Based on the experimental results, the reported TAS were enough to
achieve significant organic matter and ammonia removals. Hoang
et al. (2014) reported significant ammonia removal rate under the
same biomass concentration in the treatment of SWW at 1 °C and 20 °C.
The biofilms thickness was measured using VPSEM (Fig. 5). The
VPSEM images showed that biofilm on the bio-carrier is not uniform
and covers only the inner side of the bio-barrier. Hao et al. (2002)
showed that the attached growth nitrifying bacteria have non-uniform
properties during the treatment of wastewater treatment. The average
biofilm thicknesses were found to be in the range 69.08 to 456.11 μm
in R1, 108.44–348.69 μm in R2 and 155.09–366.19 μm in R3. Measure-
ments after 160 days showed a small decrease in the biofilm thickness
by 4.5%, 6.6% and 8.9% for R1, R2, and R3, respectively. The very small de-
crease in biofilm thickness during the 160 days of operation indicates
stable biofilm volume and density. Hao et al. (2002) showed that at
the optimal DO, the biofilm thickness ranged from 0.2 to 0.7 μm, and
this thickness was enough to contain all required organisms for high
ammonia removal. Clogging problem in R2 and R3 can be seen in the
VPSEM images R2A and R3A.
Ammonia removal with respect to biofilm volume was followed by
calculating the biofilm volume ammonia removal rate (BVAR) for the
MBBRs (R1, R2, and R3) as in Eq. (4)
SARR
BVAR ¼ ð4Þ
T bio
where Tbio is biofilm thickness. Fig. 4b displays the variation in BVAR for
R1, R2 and R3 on day 30 (T = 35 °C), 60 (T = 20 °C) and 150 (T = 4 °C).
BVAR at day 30 of operation is significantly higher than at days 60 and
150. At high temperature, the SARR was high and the biofilm thickness
was low which resulted in an increase in the BVAR values. When the op-
erational temperature decrease, both SARR and biofilm thickness de-
creased resulting in lower BVAR. The obtained results give and
information on how the SARR and biofilm changes to achieve the ex-
pected ammonia removal. MBBRs operated at low temperature showed
thicker bio-film but lower the SARR and BVRR.
Table 4 presents the Live/Dead (L/D) ratios at different depths of bio-
film and EPS analysis during the operation of the MBBRs at different
temperatures. L/D analysis revealed that MBBR R1 operated with bio-
carriers k3 have a high L/D ratio compared with the other MBBRs.
8 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425

R1-A R1-B

R2-A R2-B

R3-A R3-B

Fig. 5. VPSEM images of biofilms in R1, R2 and R3 after 80 day of operation.

 A. Ashkanani et al. / Science of the Total Environment 693 (2019) 133425 9

Table 4
Summary of live/dead test and biofilm coverage by extracellular polymeric substance for R1, R2 and R3 at different operation time.

R1 R2 R3

Time (day) Scanning depth (μm) Live (L) Dead (D) L/D % EPS (St. Dev) Live (L) Dead (D) L/D % EPS (St. Dev) Live (L) Dead (D) L/D % EPS (St. Dev)

20 0.07 0.06 1.17 0.07 69 (3.2) 0.19 0.18 1.06 71 (4.4) 0.18 0.16 1.13 73 (4.8)
0.27 0.22 1.23 0.27 0.17 0.14 1.21 0.16 0.13 1.23
1.15 0.26 4.42 1.15 5.04 2.09 2.41 4.79 1.59 3.01
0.87 0.15 5.80 0.87 0.14 0.08 1.75 0.13 0.07 1.86
0.62 0.16 3.88 0.62 0.3 0.15 2.00 0.28 0.13 2.15
40 0.07 0.063 1.11 0.07 70 (7.8) 0.19 0.18 1.06 73 (3.0) 0.18 0.16 1.13 74.9 (5.6)
0.27 0.22 1.23 0.27 0.17 0.14 1.21 0.16 0.13 1.23
1.16 0.26 4.46 1.16 5.09 1.3 3.92 4.83 1.67 2.89
0.88 0.1475 5.97 0.88 0.14 0.08 1.75 0.13 0.07 1.86
0.63 0.16 3.94 0.63 0.3 0.15 2.00 0.29 0.13 2.23
80 0.07 0.07 1.00 0.07 69 (4.1) 0.2 0.19 1.05 73 (3.8) 0.188 0.17 1.11 75 (4.3)
0.28 0.23 1.22 0.28 0.18 0.15 1.20 0.171 0.134 1.28
1.21 0.27 4.48 1.21 5.3 1.33 3.98 5.038 1.69 2.98
0.92 0.155 5.94 0.92 0.15 0.08 1.88 0.14 0.071 1.97
0.65 0.17 3.82 0.65 0.32 0.16 2.00 0.298 0.137 2.18
160 0.56 0.14 4.00 0.56 73 (3.9) 0.66 0.18 3.67 77 (4.6) 0.621 0.22 2.82 78 (4.3)
0.41 0.07 5.86 0.41 0.55 0.14 3.93 0.523 0.44 1.19
0.26 0.06 4.33 0.26 0.66 0.17 3.88 0.627 0.23 2.73
0.49 0.17 2.88 0.49 0.4 0.19 2.11 0.38 0.23 1.65
0.55 0.1 5.50 0.55 0.53 0.135 3.93 0.493 0.29 1.70

Under all scanning depths, the L/D ratio for MBBR R1 is N1.2, and the
ratio ranged from 1.2 to 6. The MBBRs R2 and R3 operated with bio-
carrier K5 and M showed L/D ratio in the range of 1.05 to 3.98 and
1.11 to 3.01, respectively. The high L/D ratio in MBBRs R1 confirms stable
and significant biological activities and healthy microorganisms. The
MBBRs R2 and R3 showed very low L/D ratio suggesting low biological
activities in the biofilm, which was observed before to have low ammo-
nia removal rates. The EPS analysis for the three reactors revealed that
the biofilm exists only on the internal surface of bio-carrier and covers
EPS at least 69% of it (Table 2). The effect of temperature on cells L/D
ratio is clear in Table 2. The L/D ratios were observed to be lower at
low temperatures.
4. Conclusion
The present study focused on the feasibility of using moving bed bio-
film reactor (MBBR) system for the treatment of ammonia-containing
effluents under real operation conditions and steady state. The novelty
of the work lies in the fact that real wastewater (effluent of secondary
treatment tanks) was used instead of synthesized wastewater input ef-
fluent. The results showed that the surface area loading rate and bio-
carrier specific surface should be considered when designing of MBBR.
The research has shown that bio-carriers with the larger surface area
have a greater propensity to become clogged under low and high load-
ing rate and this should be considered during the design of nitrifying
MBBR systems. There was significant different in ammonia removal ef-
ficiencies between bio-carriers with different surface areas as a result of
oxygen mass transfer limitation for high specific surface area bio-
carrier. Bio-carrier type has a direct effect on the kinetics of ammonia re-
moval and affects the MBBR sensitivity for changes in operational tem-
peratures. The MBBR was responsive to shock temperature in a short
time and maintained a stable and active embedded nitrifying biomass
with minimum biofilm loss or washout.
Acknowledgment
The authors gratefully acknowledge the financial support provided
by the Qatar University Internal Grant (QUUG-CENG-CHE-14\15-11).
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