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R E S E A R C H Open Access
Biological removal of phenol from saline
wastewater using a moving bed biofilm reactor
containing acclimated mixed consortia
Seyyed Ali Akbar Nakhli
1*
, Kimia Ahmadizadeh
2
, Mahmood Fereshtehnejad
1
, Mohammad Hossein Rostami
3
,
Mojtaba Safari
3
and Seyyed Mehdi Borghei
1
Abstract
In this study, the performance of an aerobic moving bed biofilm reactor (MBBR) was assessed for the removal of
phenol as the sole substrate from saline wastewater. The effect of several parameters namely inlet phenol
concentration (200–1200 mg/L), hydraulic retention time (8–24 h), inlet salt content (10–70 g/L), phenol shock
loading, hydraulic shock loading and salt shock loading on the performance of the 10 L MBBR inoculated with a
mixed culture of active biomass gradually acclimated to phenol and salt were evaluated in terms of phenol and
chemical oxygen demand (COD) removal efficiencies. The results indicated that phenol and COD removal
efficiencies are affected by HRT, phenol and salt concentration in the bioreactor saline feed. The MBBR could
remove up to 99% of phenol and COD from the feed saline wastewater at inlet phenol concentrations up to
800 mg/L, HRT of 18 h and inlet salt contents up to 40 g/L. The reactor could also resist strong shock loads.
Furthermore, measuring biological quantitative parameters indicated that the biofilm plays a main role in phenol
removal. Overall, the results of this investigation revealed that the developed MBBR system with high concentration
of the active mixed biomass can play a prominent role in order to treat saline wastewaters containing phenol in
industrial applications as a very efficient and flexible technology.
Keywords: Saline wastewater; Phenol; Biological treatment; Inhibitory effect; Acclimated biomass; MBBR
Introduction
Several industries including olive oil mills, pickled vegeta-
bles, fish processing, meat canning, dairy products, tanning
process and oil refining process generate wastewaters con-
taining high salt content and high organic concentration
(Lefebvre and Moletta 2006). Phenol and other phenolic
compounds are common organic contaminants found in
saline wastewaters formed by some of these industries such
as olive oil mills, tannery and oil refinery, ranging from
one to several hundred milligrams per liter (Moussavi et al.
2009; Edalatmanesh et al. 2008; Chiaiese et al. 2011).
Phenol has been classified as a priority hazardous or-
ganic pollutant regulated by the American Environmental
Protection Agency (EPA) (Aravindhan et al. 2009). Thus,
in order to protect human health and ecosystems from
the potential toxic effects caused by exposure to phenol,
its removal from saline wastewater with an efficient and
environmentally benign technology is quite obligatory
(Pan and Kurumada 2008). Busca et al. (2008) have re-
cently published a short review of different technologies
for phenol removal from wastewater, including physical,
chemical and biological processes. However, despite its
importance, few studies have been accomplished on phe-
nol removal from saline wastewaters.
Biological processes have advantages to physico-
chemical processes in pollution control due to their ability
to efficiently degrade the pollutants in an environmentally
sound and cost effective way (Karthik et al. 2008) and
offer efficient removal of wide range of pollutants in
wastewater treatment. Although wastewaters containing
high-concentrations of phenol are generally difficult to
treat biologically due to substrate inhibition (Ho et al.
2009); the efficient biodegradation of phenol can be
* Correspondence: a.nakhli@gmail.com
1
Biochemical and Bioenvironmental Research Center (BBRC), Department of
Chemical and Petroleum Engineering, Sharif University of Technology, PO
Box 11155-9465Azadi Ave, Tehran, Iran
Full list of author information is available at the end of the article
a SpringerOpen Journal
© 2014 Nakhli et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited.
Nakhli et al. SpringerPlus 2014, 3:112
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obtained by microbial acclimation. Phenol can be de-
graded by pure cultures as well as mixed bacterial con-
sortia (Bajaj et al. 2008). In addition, biological
treatment of saline wastewater usually results in low re-
moval efficiencies because of the adverse effects of salt
on microbial flora (Uygur 2006), but by a proper adap-
tation of the biomass to a desired salt concentration or
use of halophilic microorganisms, the detrimental ef-
fects of salinity on the overall bioprocess performance
can be also mitigated (Aloui et al. 2009; Moussavi et al.
2010). Among the bioprocesses invented for biological
treatment of wastewater, bioreactors implying biofilm sys-
tems play important roles in the detoxification of hazard-
ous organic contaminants such as phenol (Hosseini and
Borghei 2005). Moving bed biofilm reactor (MBBR) is a
highly effective biological treatment process that was in-
troduced about 30 years ago and now it is used in large-
scale all over the world (Rusten et al. 2006). MBBR is a
completely mixed and continuously operated biofilm re-
actor that is designed to offer the positive aspects of bio-
film process including a stable removal efficiency of
toxic pollutants, compact and simplicity of operation;
without its drawbacks including high head loss,
medium channeling and clogging (Chen et al. 2008;
Delnavaz et al. 2010). In addition, moving bed reactors
provide a better control of biofilm thickness and higher
mass transfer characteristics (Moussavi et al. 2009).
The concentration of biomass in MBBR can be in-
creased either by raising the amount of moving media
(Bassin et al. 2011), or using media with a high effective
biofilm surface area, that enhances resistance to toxicity
and consequently improves MBBR performance. As a
consequence of such advantages MBBR process has
been recently used for the removal of many toxic
wastewaters including landfill leachate (Chen et al.
2008), aniline (Delnavaz et al. 2010), ammonium from
saline wastewater (Bassin et al. 2011), coal gasification
wastewater (Li et al. 2011), thiocyanate (Jeong and Chung
2006) and antibiotic fermentation-based pharmaceutical
wastewater (Xing et al. 2013).
Treatment of phenol-laden saline wastewater using
MBBR has not been reported yet. Reported applications
that have dealt with biological process are mostly limited
to the cases of single microbial species or low inlet phenol
concentrations (Dosta et al. 2011; Afzal et al. 2007; Leitão
et al. 2007; Kobayashi et al. 2007); both of which may have
limitations in the field application that contaminant con-
centrations of targeted wastewater may alter from low to
high. Accordingly, the basic purpose of this research was
to investigate the performance of an aerobic MBBR to
examine the above-mentioned benefits in treating syn-
thetic phenol-laden saline wastewater using a mixed cul-
ture that gradually acclimated to phenol and salt. To
achieve this aim, the MBBR was operated at different op-
erational conditions including inlet phenol concentration,
hydraulic retention time (HRT), inlet salt content and
shock loadings.
Materials and methods
MBBR experimental setup
The study was performed using the cylindrical MBBR re-
actor (see Figure 1 for more details), made from Plexiglas
with internal diameter, height and wall thickness of 14, 75
and 0.5 cm respectively, equivalent to 11.5 L total volume.
The effective depth of wastewater in the reactor was
65 cm (10 L working volume) filled with up to 50% the
floating biofilm carrier elements composed of high density
polyethylene (HDPE) with a density of 0.96 g/cm
3
and an
effective surface area of 520 m
2
/m
3
. The aeration system
was proceeded with the aid of central compressed air
Figure 1 Flow diagram of MBBR experimental setup.
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system. Fine bubbles were produced by the aeration sys-
tem. These bubbles could provide a sufficient mixing to
keep the carriers moving in the reactor. In order to keep the
carriers in the reactor, outlet diameter was designed to be
smaller than carrier’s size. Synthetic wastewater was injected
to the reactor by a peristaltic pump with a flow controlling
mechanism. Pumping rate of the wastewater into the re-
actor was regulated according to the working volume and
HRT. In order to take samples and monitor the perform-
ance of the system in phenol and chemical oxygen demand
(COD) removal, two sampling ports were provided on the
influent and effluent lines of the reactor.
MBBR operation procedure
The MBBR used in this investigation was operated at
continuous mode (except in biomass acclimation phase)
for 251 days. Different phases of the experiment and the
range of investigated variables are presented in Table 1.
In continuous system, the MBBR was run to investigate
the effects of different operational variables on phenol and
COD removal at the steady state operational conditions;
which it was assumed that the steady state condition oc-
curred when changing in the removal efficiency was
within ±5% for consecutive HRTs at each operational run.
The reactor was operated at room temperature (23 ± 2 ºC)
under dissolved oxygen (DO) concentration of 4–5mg
O
2
/L controlled by regulating the aeration rate.
Wastewater and inoculum preparation
Synthetic wastewater was prepared daily by adding phe-
nol, nutrient stock solution and NaCl to tap water. Phenol
was the sole carbon and energy source for the biomass in
the MBBR. The nutrient solution consisted of Urea as a
nitrogen source, (NH4)3PO
4
.3H
2
O as a nitrogen and
phosphorus source and the trace elements. The COD:N:P
ratio in the feed wastewater was kept at 100:5:1 through-
out the experiment, where 1 mg/L of phenol is equal to
2.15 mg/L of COD. All chemicals were of analytical grade
except for NaCl, which was purchased commercially. In
addition, the pH of the inlet wastewater was kept at
neutral range.
During the start-up, the MBBR was inoculated with an
activated sludge obtained from Pars oil wastewater treat-
ment plant. The health of the activated sludge was verified
by microscopic study. Acclimation of the activated sludge
to phenol and salt was lasted 90 days and was performed
in the batch system. First, phenol concentration in the re-
actor was increased step-wise up to 500 mg/L, and then
salt content in the reactor was increased step-wise up to
30 g/L. Phenol concentration remained at 500 mg/L dur-
ing this stage. In each phenol and salt concentration, the
reactor was operated until the removal efficiency of phe-
nol exceeded 90% after passing 1 day. During this phase,
the biofilm was gradually formed on the carriers. Micro-
scopic observations revealed that the active and enriched
salt-tolerant phenol-degrading biofilm was achieved. This
biomass was used as an inoculum to the reactor.
Analytical method
To evaluate the performance of the MBBR, samples from
inlet and outlet of the reactor was taken and analyzed at
HRT interval. The measured parameters in inlet samples
were phenol, COD, chloride, ammonia nitrogen, phos-
phate and pH; whereas phenol, COD and chloride were
measured in outlet samples. The parameters of pH, DO
and temperature of the mixed liquor were daily mea-
sured in order to control the optimum condition for
bacterial growth in the reactor. For evaluating biomass
characterization the parameters of mixed liquor sus-
pended solid (MLSS), biofilm solid (BS), biofilm thick-
ness and specific oxygen uptake rate (SOUR) were
measured in the mixed liquor and the carriers samples
routinely. In order to measure phenol and COD, the
samples were filtered through a filter with 0.45 mm
pore size before analysis. Phenol concentrations were
measured spectrophotometrically, using a Unico-UV
9200 UV/VIS Spectrophotometer by the colorimetric 4-
aminoantipyrine according to the procedure given in
the Standard Methods (APHA 2005). The pH, DO and
temperature were measured using specific electrodes.
The parameters of chloride, ammonia nitrogen, phos-
phorous, and MLSS were determined according to the
Table 1 Experimental phases and MBBR operation timing schedule
Phase Day Operation Inlet concentration, Cin (mg/L) Salt content (g/L) HRT (h)
Phenol COD
10–90 Biomass acclimation 50–500 107.5–1075 0–30 -
291–150 Effect of C
in
200–1200 430–2580 30 24
3 151–190 Effect of HRT 800 1720 30 8–24
4 191–245 Effect of salt content 800 1720 10–70 18
5 246–247 Response to organic shock loading - - 30 18
6 248–249 Response to hydraulic shock loading 800 1720 30 -
7 250–251 Response to salt shock loading 800 1720 - 18
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Standard Methods (APHA 2005). The Parameters of COD
for saline samples, BS, biofilm thickness and SOUR were
determined according to the procedures used by Vyrides
and Stuckey (2009); Plattes et al. (2006); Horn et al. (2003)
and Moussavi et al. (2009), respectively. The morphology
of the biomass was visualized using a microscope with
1000× magnification factor.
Results and discussion
Effect of inlet phenol concentration on removal efficiency
The effect of inlet phenol concentration ranging from 200
to 1200 mg/L (430–2580 mg COD/L) on the performance
of the MBBR in phenol and COD removal efficiency was
assessed over six runs under the conditions given in
Table 1. Figure 2 depicts average phenol and COD re-
moval efficiency resulted from defined steady-state condi-
tions versus inlet phenol concentration.
As demonstrated in Figure 2 increasing inlet concentra-
tion up to 800 mg/L did not significantly affect the per-
formance of the MBBR in phenol and COD removal and
the efficiencies were over 99% for both parameters. This
denotes that the part of phenol metabolized as a carbon
and energy source has been completely biodegraded, al-
though further increasing inlet concentration showed an
adverse effect on the removal efficiency. Particularly, in-
creasing inlet concentration to 1000 and 1200 mg/L re-
sulted in decreasing phenol removal below 97.6% and 94%,
respectively. Rate of decreasing COD removal was higher
than that of phenol and was below 93% and 85.1% at inlet
concentration of 1000 and 1200 mg/L, respectively.
The results might be explained by consideration that at
low phenol concentrations no effect is noted on gross mea-
sures of metabolic activity such as specific growth rate, res-
piration rate, rate of synthesis, etc. By increasing phenol
concentration, the biological parameters will increase due
to stimulation of metabolism of the microorganisms. Even-
tually, the concentration is reached to a point which fur-
ther increase of the concentration does not increase the
biological parameters. Further increasing phenol concen-
tration will eventually cause the physiological parameters
decrease and the substrate utilization inhibition to occur
(Hosseini and Borghei 2005).
Because phenol was the sole substrate the difference be-
tween COD equivalent of measured phenol and COD mea-
sured in the effluent could be explained by accumulation of
organic intermediates (metabolites) that were generated dur-
ing the partially phenol biodegradation caused by inhibitory
effect of high phenol concentration in synthetic saline waste-
water on microbial activities (Moussavi et al. 2010). The con-
centration of metabolites at inlet concentration of 1000 and
1200 mg/L was 97.3 and 230.6 mg/L as COD, respectively.
The results revealed that at phenol concentrations less
than 800 mg/L, a complete mineralization occurred and
no metabolites were generated under the given conditions
of operation. Thus, this value was selected as an optimum
inlet phenol concentration for the following phases of the
experiment. Therefore the optimum surface loading rate
based on inlet concentration (at HRT of 24 h) on the
MBBR was found to be 3.08 g phenol/m
2
.day (6.62 g
COD/m
2
.day). Accordingly, the MBBR could effectively
remove both phenol and its COD from the synthetic sa-
line wastewater.
These kinds of behavior and conclusions have also been
shown by other researchers, although by using a pure cul-
ture (Afzal et al. 2007; Leitão et al. 2007; Kobayashi et al.
2007) or a phenol degrading mixed culture (Moussavi
et al. 2010) that cannot be applicable in industrial scale.
High capacity of the investigated MBBR to complete re-
moval of phenol in saline wastewater could be attributed
to use of the mixed culture of gradually acclimated active
85
90
95
100
200 400 600 800 1000 1200
Removal Efficiency (%)
Inlet Phenol Concentration (mg/L)
Phenol Removal Efficiency
COD Removal Efficiency
Figure 2 Phenol and COD removal efficiencies versus inlet phenol concentrations at HRT of 24 h and salt content of 30 g/L.
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biomass to phenol and salt, using the biofilm carriers with
high specific surface area available for microbial growth
and high filling ratio.
Effect of hydraulic retention time on removal efficiency
In order to determine the required retention time for the
efficient removal, which specifies the size of facilities in
biological wastewater processes, the next phase of the ex-
periment was designed to assess the effect of various HRT
of 24, 20, 18, 16, 12 and 8 h on the performance of the
MBBR in phenol and COD removal under the operating
conditions given in Table 1. The reactor was operated dur-
ing each HRT until defined steady-state conditions were
attained. The mean phenol and COD removal efficiencies
versus HRTare demonstrated in Figure 3.
Figure 3 shows that phenol and COD removal efficien-
cies were not affected by reducing HRT down to 18 h and
the removal efficiencies of both parameters were greater
than 99%. Although by further reducing HRT, the removal
efficiencies of both phenol and COD were reduced and at
higher values of HRT, the investigated MBBR was less
sensitive to reduction of HRT. By reducing HRT to 16, 12
and 8 h, the mean removal efficiency of phenol decreased
to 98.9%, 93.9% and 84.4%, respectively. COD removal
efficiency decreased with higher rate in comparison to
that of phenol and at HRT of 16, 12 and 8 h the mean
removal efficiency of COD was below 97.8%, 89.2% and
74.4%, respectively.
This behavior might be explained by considering that
the decrease of HRT until the retention time is enough
for complete oxidation, has no remarkable effect on the
removal efficiency. Further decreasing HRT leads to in-
complete degradation. In addition, increase in hydraulic
load speeds leads to detachment of the biofilm from the
carrier elements and reduction of active biomass in the
reactor (Hosseini and Borghei 2005).
Reducing HRT and consequently increasing phenol
loading rate over the biodegradation capacity of the biomass
in the reactor might inhibit the complete mineralization re-
sulted in increasing metabolites concentration in the efflu-
ent. There was no considerable accumulation of metabolites
down to retention time of 18 h. By reducing HRT to16, 12
and 8 h, phenol inhibition occurred and metabolites
concentration in the effluent was increased to 18.8, 80
and 171.2 mg COD/L, respectively.
It can be concluded from above that an optimum HRT
for the MBBR under the selected operational conditions
was 18 h, at which phenol and COD removal efficiencies
were above 99% and no metabolites were detected. Thus,
this value was selected as the optimum retention time for
the next phases of the experiment. Accordingly, the
optimum surface loading rate based on HRT (at inlet phe-
nol concentration of 800 mg/L) on the MBBR was found
to be 4.1 g phenol/m
2
.day (8.82 g COD/m
2
.day). These re-
sults indicate that the MBBR inoculated with the active
mixed biomass adapted to phenol and salt can efficiently
remove high phenol loading rate and associated COD.
The adverse effect of HRT on COD removal efficiency in
the MBBR system has also reported by other researchers (Li
et al. 2011; Hosseini and Borghei 2005). Based on the avail-
able literature, no experiments were found dealing with re-
moval of phenol from saline wastewater by using mixed
active cultures adapted to phenol and salt in the MBBR. In
comparison to other bioreactors, the investigated MBBR indi-
cated a high performance in the removal of phenol and COD
from saline wastewater. Moussavi et al. (2010) worked on
phenol removal from saline wastewater with a granular se-
quencing batch reactor (GSBR) containing phenol-degrading
consortia adapted to salt under operational conditions of
70
75
80
85
90
95
100
812162024
Removal Efficiency (%)
HRT (hr)
Phenol Removal Efficiency
COD Removal Efficiency
Figure 3 Profile of phenol and COD removal efficiencies versus HRT at optimum phenol concentration of 800 mg/L and salt content of 30 g/L.
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cycle time of 17 h and inlet phenol concentration of
1000 mg/L, finding 99% removal efficiency. Dosta et al.
(2011) evaluated the performance of a membrane biological
reactor (MBR) for removing phenol from saline wastewater
at HRT of 12–17 h, inlet phenol concentration of 8–15 mg/L
and reported COD removal efficiency of over 98.5%. The
great performance of the MBBR in this study could be espe-
cially due to the existence of a high concentration of the ac-
climated and active mixed culture of biomass and using a
high percentage occupation of the carriers with a high effect-
ive surface area.
Effect of salt content on removal efficiency
In this phase of the experiment, the effect of salt content
of synthetic saline wastewater ranging from 10 g/L to
70 g/L was assessed on the behavior of the MBBR under
the previously optimized conditions given in Table 1. The
MBBR was operated at each salt concentration until deter-
mined steady-state condition was achieved. The mean
phenol and COD removal efficiencies as a function of salt
concentration in the feed stream are demonstrated in
Figure 4.
According to Figure 4 inlet salt content in the range of
10–50 g/L had negligible effect on the performance of the
MBBR in phenol removal and the efficiency remained
greater than 99%. However, further increase in salt con-
centration to 60 g/L and subsequently to 70 g/L, resulted
in decrease in phenol removal efficiency down to 98.1%
and 96.7%, respectively. The effect of salt content up to
40 g/L on COD removal was insignificant and the effi-
ciency remained around 99%. However, when salt content
was increased to 50, 60 and 70 g/L, COD removal
90
92
94
96
98
100
10 20 30 40 50 60 70
Removal Efficiency (%)
Salt Content (g/L)
Phenol Removal Efficiency
COD Removal Efficiency
Figure 4 Average of phenol and COD removal efficiencies versus inlet salt content at optimum condition of phenol concentration and HRT.
0
200
400
600
800
1000
1200
1400
0
100
200
300
400
500
012345678910
Outlet Total& Phenolic COD Concentration
(mg/L)
Time (hr)
Inlet Phenol Concentration
(mg/L)
Outlet Phenolic COD
Outlet Total COD
Inlet Phenol
Figure 5 Variation of outlet total and phenolic COD concentration during phenol shock load at the HRT of 18 h and salt content of 30 g/L.
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efficiency was reduced down to 97.4%, 95.3% and 92.7%,
respectively.
Gradually acclimation of the biomass to specific salt
concentration can mitigate the detrimental effect of salin-
ity on microbial activity. Much more salt content causes
disintegration of cells because of the loss of cellular water
(plasmolysis) or the recession of the cytoplasm which is
induced by an osmotic difference across the cell wall and
cause of outward flow of intracellular water resulting in
the loss of microbial activity and cell dehydration (Abou-
Elela et al. 2010).
By decreasing microbial activity in high salt content,
metabolites concentration was increased to 9.6, 31, 47.5
and 68.2 mg COD/L in salt content of 40, 50, 60 and
70 g/L, respectively.
Hinteregger and Streichsbier (1997) worked on the ef-
fect of salt content (1–14%) on biotreatment of saline
phenolic wastewater by a moderately halophilic strain and
showed the adverse effect of salt on biotreatment. None-
theless, Moussavi et al. (2010) showed that salt content in
the range of 3–8% has no effect on the GSBR performance
containing the phenol-degraded biomass adapted to salt.
Stability of the MBBR against high salt content in the
range of 10–40 g/L can be related to establishing the bio-
mass containing a high concentration of salt-adapted mi-
croorganisms. It can be inferred from above that the
operating MBBR with the mixed consortia acclimated bio-
mass can attain a high performance for phenol-laden sa-
line wastewater in terms of phenol and COD removal
under the different operational conditions.
0
9
18
0
100
200
300
400
500
600
012345678910
Outlet Total& Phenolic COD Concentration
(mg/L)
Time (hr)
HRT (hr)
Outlet Phenolic COD
Outlet Total COD
HRT
Figure 6 The effect of hydraulic shock load on the performance of the MBBR at inlet phenol concentration of 800 mg/L and salt content of 30 g/L.
0
10
20
30
40
50
60
70
80
0
20
40
60
80
100
012345678910
Outlet Total& Phenolic COD Concentration
(mg/L)
Time (hr)
Inlet Salt Content (g/L)
Outlet Phenolic COD
Outlet Total COD
Inlet Salt Content
Figure 7 Effect of salt shock load on outlet total and phenolic COD at inlet phenol concentration of 800 mg/L and HRT of 18 h.
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Response to shock loading
Shock loading can be applied by sudden increase of or-
ganic concentration, flow rate and salt content in saline
wastewater. Therefore in this phase of the study, response
of the investigated MBBR to mentioned shock loads was
evaluated.
Response to organic shock loading
In order to evaluate the adverse effect of phenol shock
load on the MBBR performance, a sudden increase of inlet
phenol concentration from 800 to 1400 mg/L was applied
to the bioreactor for a period of 4 h under the conditions
presented in Table 1. During this period and after that the
concentrations of outlet phenol and COD were monitored
every 1 h until the steady state condition was re-
established, as shown in Figure 5. It can be seen that out-
let phenolic and total COD were increased from 10 and
15 mg/L to the maximum concentrations of 68.4 and
145 mg/L, respectively. Around 5 h after shock load outlet
phenolic and total COD gradually reached to the near
steady state values of 10.75 and 25 mg/L, respectively.
Response to hydraulic shock loading
In order to study the reactor stability against a sudden
variation of flow rate, HRT was changed from 18 to 9 h
for a period of 4 h under the conditions listed in Table 1.
To understand the trend of outlet phenolic and total COD
concentration during shock loading and after that, the ef-
fluent was sampled and analyzed at 1-h-intervals and the
results are shown in Figure 6. According to Figure 6 outlet
Table 2 Characteristics of biomass during study
Parameter Unit Value
Mixed liquor suspended solid mg/L 250–640
Biofilm solid mg/L 1405–4450
Biofilm thickness μm18–58
Specific oxygen uptake rate mg O
2
/mg VSS.d 0.62 ± 0.07
Figure 8 Photomicrograph of biomass. (a) biofilm (b) mixed liquor.
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phenolic and total COD started to increase from 10 and
15 mg/L to the maximum concentrations of 138 and
235 mg/L, respectively; then gradually started to decrease
to the near steady state value of 18.7 and 37.5 mg/L about
5 h after shock load.
Response to salt shock loading
To evaluate the resistance of the MBBR against jump of
inlet salt concentration, a sudden change was applied to
the reactor where inlet salt concentration increased from
30 to 80 g/L for a period of 4 h under the conditions
listed in Table 1. The changes in outlet phenolic and total
COD concentration under this condition and after that
were monitored hourly and the results are presented in
Figure 7. Figure 7 depicts that outlet phenolic and total
COD concentrations changed insignificantly and were in-
creased from 10 and 15 mg/L to the maximum concentra-
tions of 10.3 and 20 mg/L, respectively. But after
switching the influent to the initial condition, the effluent
was remained unchanged, which could be because of
remaining a high salt content in the bioreactor.
It can be inferred from the above results that the MBBR
exhibited a high stability against organic, hydraulic and
salt shock loadings and recovered from these changes in a
relatively short time. This low sensitivity to shock loadings
could be due to the existence of a high concentration of
biomass containing gradually acclimated and active micro-
bial consortia and a high filling ratio of the biofilm carriers
with a high effective surface area in the reactor. High sta-
bility of the MBBR against shock loadings had previously
reported by other researchers (Chen et al. 2008; Hosseini
and Borghei 2005). These advantages introduce the MBBR
as an effective and stable process for the removal of phe-
nol from saline wastewaters.
Biomass characteristics
The biomass characteristics were evaluated both in suspen-
sion and biofilm during this study. The determined charac-
teristics were MLSS, BS, biofilm thickness and SOUR. The
range of measured biological parameters during this study
is presented in Table 2. In the continuous system, the sus-
pended biomass in the bioreactor was negligible in com-
parison to the biofilm attached to the carriers. It can be
inferred that the attached biomass had the main role in the
removal of phenol and COD rather than the suspended
biomass in the investigated MBBR. The thickness of the
biofilm formed on the media was in the range of effective
biofilm thickness (the depth of the biofilm to which the
substrates have penetrated) (Rusten et al. 2006). The SOUR
values indicate a high activity of the biomass in the reactor
which could be attributed to the moving media containing
a thin biofilm that improves the oxygen and substrate
transfer rate and contact between the substrate and the
biomass, therefore, enhances degradation rate (Moussavi
et al. 2009).
Microscopic examinations were carried out in order to
observe the existing microorganisms in the bioreactor dur-
ing the experiments. Photomicrograph of the biofilm and
the mixed liquor are demonstrated in Figure 8. As shown
in Figure 8(a) predominant species in the biofilm was yeast
and some mold and bacteria also were found in the bio-
film, but there was no indication of these groups of micro-
organisms in the liquid bulk where bacteria was the main,
as shown in Figure 8(B). As demonstrated in Figure 8(A),
the existence of yeast ongoing to budding and fission im-
plied a high activity of the biofilm inside of the bioreactor.
Dan et al. (2003) indicated that yeast culture is more effi-
cient in treating high organic–high salinity wastewater
compared to bacterial cultures. Hence, the high perform-
ance of the investigated MBBR in phenol removal effi-
ciency could be attributed to the existence of the high
concentration of yeast in the biofilm.
Conclusion
The present work investigated the performance of a bench
scale MBBR for phenol removal from saline wastewater.
The results revealed that the MBBR provides improved
phenol and COD removal efficiencies. Inlet phenol con-
centrations up to 800 mg/L did not significantly affect the
performance of the MBBR with HRT of 24 h and salt con-
tent of 30 g/L, where phenol and COD removal efficiencies
were above 99%. Optimum HRT for the reactor was 18 h,
such that decreasing HRT below this value led to reduction
of the removal efficiencies of both phenol and COD. The
MBBR exhibited low sensitivity to increasing salt concen-
trations up to 40 g/L. The reactor was very stable against
phenol, hydraulic and salt shock loadings and performed
well under various operational conditions. The active bio-
film containing yeast as a predominant species performed
themainroleinphenolremovalintheMBBR.Overall,
high performance of the investigated MBBR in the removal
of phenol from saline wastewater could be attributed to ex-
istence of the mixed culture of gradually acclimated bio-
mass to phenol and salt and using a high filling ratio of the
biofilm carriers with a high effective surface area.
Abbreviations
MBBR: Moving bed biofilm reactor; COD: Chemical oxygen demand;
HRT: Hydraulic retention time; EPA: Environmental protection agency;
HDPE: High density polyethylene; DO: Dissolved oxygen; MLSS: Mixed liquor
suspended solid; BS: Biofilm solid; SOUR: Specific oxygen uptake rate;
GSBR: Granular sequencing batch reactor; MBR: Membrane biological reactor.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
SAAN and KA designed and performed the experiments, analyzed the data
and wrote the paper; MF and MHR performed the experiments and
prepared the final manuscript; MS performed the experiments and revised
Nakhli et al. SpringerPlus 2014, 3:112 Page 9 of 10
http://www.springerplus.com/content/3/1/112
the manuscript; SMB gave technical support and conceptual advice. All
authors read and approved the final manuscript.
Acknowledgement
The authors wish to thank Biochemical and Bioenvironmental Research
Center, Sharif University of Technology for their support and provision of
funds to carry out this research.
Author details
1
Biochemical and Bioenvironmental Research Center (BBRC), Department of
Chemical and Petroleum Engineering, Sharif University of Technology, PO
Box 11155-9465Azadi Ave, Tehran, Iran.
2
Department of Biological Sciences,
Shahid Beheshti University, PO Box 19839-63113Velenjak, Tehran, Iran.
3
Department of Chemical Engineering, Amirkabir University of Technology,
PO Box 15875-4413, Tehran, Iran.
Received: 27 September 2013 Accepted: 9 December 2013
Published: 26 February 2014
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Cite this article as: Nakhli et al.:Biological removal of phenol from saline
wastewater using a moving bed biofilm reactor containing acclimated
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