Journal of Hazardous Materials 323 (2017) 311–318

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Journal of Hazardous Materials

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

Enhanced biodegradation of phenolic compounds in landfill leachate

by enriched nitrifying membrane bioreactor sludge
Varinthorn Boonyaroj a , Chart Chiemchaisri b,∗ , Wilai Chiemchaisri b , Kazuo Yamamoto c
a
Department of Environmental Science and Natural Resources, Faculty of Science and Technology, Rajamangala University of Technology Phra Nakhon,
Bangkok 10800, Thailand
b
Department of Environmental Engineering & Center for Advanced Studies in Industrial Technology, Faculty of Engineering, Kasetsart University, Bangkok
10900, Thailand
c
Environmental Science Center, University of Tokyo, Tokyo 113, Japan

h i g h l i g h t s

• BPA and BHT removals was investigated in two-stage MBR over 300 days.
• Microbial community for degrading compounds were established under long SRT.
• Target compounds were removed at 65–72% at high influent concentration of 1 mg/L.
• Most of the compounds were biodegraded in aerobic reactor.
• Nitrifying enriched sludge enhanced BPA and BHT degradation kinetics by 44%.

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

Article history: The role of autotrophic nitrification on the biodegradation of toxic organic micro-pollutants presented
Received 31 January 2016 in landfill leachate was assessed. A two-stage MBR system consisting of an inclined tube incorporated
Received in revised form 13 May 2016 anoxic reactor followed by aerobic submerged membrane reactor was operated under long sludge age
Accepted 30 June 2016
condition in which nitrifying bacteria could be enriched. During the reactor operation, organic removal
Available online 1 July 2016
efficiencies were more than 90% whereas phenolic compounds including bisphenol A (BPA) and 4-methyl-
2,6-di-tert-butylphenol (BHT) were removed by 65 and 70% mainly through biodegradation in the aerobic
Keywords:
reactor even at high feed concentrations of 1000 ␮g/L for both compounds. Batch experiments revealed
Emerging contaminant
Enriched nitrifying sludge
that enriched nitrifying sludge with nitrifying activities could biodegraded 88 and 75% of BPA and BHT,
Landfill leachate largely improved from non-nitrifying sludge and enriched nitrifying sludge with the presence of inhibitor.
Membrane bioreactor The first-order kinetic rates of BHT and BPA removal were 0.0108 and 0.096 h−1 , also enhanced by 44%
Phenolic compounds from the non-nitrifying sludge.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction natural environment even when present at low concentration and

Typical municipal landfill leachate contain several groups of pol-
lutants including organic matter, nutrients, inorganic pollutants
such as heavy metals and, toxic organic micro-pollutants including
xenobiotic organic compounds or XOCs [1]. XOCs that are found
in landfill leachate are usually difficult to be removed by conven-
tional leachate treatment system due to their complex molecular
structures. The XOCs are harmful to the ecosystem and the
∗ Corresponding author.
E-mail addresses: varinthorn.b@rmutp.ac.th (V. Boonyaroj), fengccc@ku.ac.th,
c.chiemchaisri@gmail.com (C. Chiemchaisri), fengwlc@ku.ac.th (W. Chiemchaisri),
yamamoto@esc.u-tokyo.ac.jp (K. Yamamoto).
they are generally not being regulated [2]. Among them, phenolic
compounds and phthalic acid esters (PAEs) are commonly found
in landfill leachate [3–5]. Several phenolic compounds and PAEs
are hydrophobic in nature and they can be removed by sorption
onto sludge and being removed from water during solid-liquid
separation. Meanwhile, the hydrophilic compounds, if not prop-
erly biodegraded in the treatment processes, could remain in the
final effluent and being discharged into the environment. Bisphenol
A (BPA) and 4-Methyl-2,6-di-tert-butylphenol (BHT) are the phe-
nolic compounds being focus in this research as they presented at
higher concentrations (up to several hundreds ␮g/l level) in munic-
ipal landfill leachate in Thailand and they are detected mainly in
water soluble form [6]. BPA is a monomer used for polycarbonate

http://dx.doi.org/10.1016/j.jhazmat.2016.06.064
0304-3894/© 2016 Elsevier B.V. All rights reserved.
312 V. Boonyaroj et al. / Journal of Hazardous Materials 323 (2017) 311–318
and epoxy resins. BHT is a widely used synthetic antioxidant found
in all types of manufactured items, from foodstuffs to cosmetics,
rubber, and paint. DEHP is a plasticizer used to enhance softness
and flexibility in PVC products. There is an urgent need to remove
these substances from landfill leachate during the treatment in
order to reduce their adverse effect on the natural ecosystem and
the environment.
Several treatment processes have been applied to the treat-
ment of landfill leachate including membrane bioreactor (MBR) [7].
MBR is an advanced wastewater treatment process for wastew-
ater containing high organic, and nutrient loading, and is found
to be effective in the removal of some toxic organic micro-
pollutants [8,9]. In previous research, it was reported that toxic
organic micro-pollutants could be removed to a greater extent
using MBRs than conventional treatment process [10]. In MBR,
prolonged sludge retention time (SRT) and nitrifying condition
were found to improve micro-pollutant removal during biological
treatment using MBR [11]. For hydrophilic compounds, its sorp-
tion capacity to solids is limited and complete removal can only
be achieved through their biodegradation, For hydrophobic com-
pounds, sorption to the biomass and subsequent retention of the
solids by the membrane filtration are the main removal mecha-
nisms [12,13]. Several operational conditions employed in MBRs
favor and enhance biotransformation and mineralization of micro-
pollutants. MBRs can be operated under long SRT condition as its
operation does not depend on sludge settling ability. Long SRT
operation allows slow growing microorganisms to adapt which
results in high diversity of microbial community including nitrify-
ing bacteria in the system [14]. Higher biomass concentrations also
lead to intensification of biological processes and may increase the
interaction between microorganisms and more likely for genetic
information exchange to occur. Under higher biomass concentra-
tions, the food to microorganism (F/M) ratio is also lower which
could result in more complete mineralization.
Two-stage MBR utilizing inclined-plate separator in first stage
anoxic reactor followed by second stage aerobic reactor with sub-
merged membrane module for solid-liquid separation has been
found effective for the treatment of wastewater with stable effi-
ciency [15]. Similar system was also found suitable to the treatment
of landfill leachate [6,16]. During the treatment of landfill leachate,
high removal efficiencies of phenolic compounds and PAEs more
than 95% were observed and the main mechanisms responsible
for their removals was biodegradation under aerobic condition
[6]. In mixed culture aerobic sludge system, heterotrophic and
autotrophic microorganisms are responsible for the biodegrada-
tion of toxic organic micro-pollutants presented in wastewater
through their metabolic and co-metabolic activities respectively
[17]. The presence of autotrophic nitrifying organisms which are
cultivated under long sludge age condition in biological wastewater
treatment are found beneficial for biodegradation of toxic organic
micro-pollutants [18,19]. Nevertheless, its capacity in removing
micro-pollutants from real landfill leachate was still not clear espe-
cially if the micro-pollutants are presented at higher concentration
up to mg/l level. In this aspect, the unique sludge characteristics
cultivated in MBR under high biomass concentration and long SRT
condition through the operation without excess sludge wastage
could also enhance to the removals of those compounds. This study
aims at determination on the role of nitrifying activity of sludge
in biodegradation of phenolic compounds, i.e. BPA and BHT by
enriched nitrifying sludge of the MBR system. In this research, the
treatment performance and removal efficiencies of toxic organic
micro-pollutant in MBR was investigated. Batch experiments using
cultivated sludge obtained from MBR under stable operating con-
dition was then carried out to investigate maximum capacity for
biodegradation of studied compounds under the presence and
absence of nitrifying activities. The information derived from this
Fig. 1. Schematic of two-stage MBR.
research will be beneficial for the optimization of MBR operation to
achieve effective removals of toxic organic micro-pollutants from
landfill leachate.
2. Materials and methods
2.1. Operation of MBR for cultivation of enriched nitrifying sludge
2.1.1. Reactor set-up and operating condition
Laboratory-scale MBR unit with a treatment capacity of 60 L/d
was used in this study. The schematic diagram of the MBR is illus-
trated in Fig. 1. The anoxic reactor of 0.3 m width, 0.30 m length,
and 0.5 m height (30 L volume) was used. An inclined tube mod-
ule (2.5 cm channel width and 30 cm depth) was installed in the
tank for sludge separation. In an aerobic tank of identical size as
the anoxic tank, a Sterapore SADFTM (SADF0790 M mini module)
PVDF hollow fiber membrane module (Mitsubishi Rayon Company,
Japan) was submerged for solid liquid separation. A membrane
had nominal pore size of 0.4 ␮m with total effective membrane
surface area of 0.07 m2 surface area. Intermittent suction (5 min
on and 1 min off) was performed to withdraw permeate from the
membrane module at constant rate of 60 L/d. The aeration system
was used to continuously supplied oxygen to the aerobic reac-
tor to maintain the dissolved oxygen (DO) level at 3–4 mg/L. The
MLSS concentration in aerobic tank was maintained at 10,000 to
12,000 mg/L in order to control membrane fouling by perform-
ing sludge recirculation from the aerobic tank back to anoxic tank
at 100% of feed flow rate. Sludge wastage was not performed
except for analysis purposes (approx. 12 mL every 2 days inter-
val). Hydraulic retention time (HRT) in both tanks was kept at
12 h. The average membrane permeate flux was controlled at
0.4286 m3 /m2 d.
Influent leachate representing a typical situation in Thailand
where fresh juice from solid waste collection trucks and leach-
ing liquid from operating and closed landfill area are collected and
stored together in storage pond awaiting their treatment. To ensure
consistency in their characteristics for reactor experiment, fresh
juice and landfill leachate collected separately from trucks and
landfill area respectively was mixed at 1:10 mixing ratio (v/v) and
used as feeding leachate. This preparation was performed to ensure
that the organic concentrations were kept relatively constant and
not significantly fluctuated along the experimental period. During
the experiment, raw and treated wastewater characteristics were
regularly monitored on a weekly basis.
Leachate samples were kept inside glass containers and stored
at a temperature of 4 ◦ C. Prior to analysis, the wastewater samples
were filtered through the glass microfiber filter (GF/C). Biomass
concentration and characteristics in terms of mixed liquor sus-
pended solids (MLSS), sludge volume index (SVI), particle size
analysis (Mastersizer 2000E, Malvern, UK), and extracellular poly-
meric substances (EPS) production using lowry method, and
phenol/sulfuric acid method were occasionally determined. The
characteristics of landfill leachate were performed according to
 V. Boonyaroj et al. / Journal of Hazardous Materials 323 (2017) 311–318
Table 1
Feeding leachate characteristics of two-stage MBR.
Parameter MBR influent
Range
Temp(◦ C) 27.5–30.7
pH 8.63–8.89
BOD(mgL−1 ) 6000–6987
COD(mgL−1 ) 9000–9846
TOC(mgL−1 ) 2000–2882
SS(mgL−1 ) 1120–1680
TKN(mgL−1 ) 200–230
NH4 + -N(mgL−1 ) 112–128
NO3 − -N(mgL−1 ) 1.215–2.148
NO2 − -N(mgL−1 ) 0.010–0.125
EC(mS(cm)−1 ) 23.0–30.1
Standard Methods for the Examination of Water and Wastewa-
ter [20]. pH and electrical conductivity (EC) were analyzed using
portable meters (Digicon). Organic compounds were determined
in terms of biochemical oxygen demand (BOD5 ) using a 5-day
BOD test, chemical oxygen demand (COD) using closed dichromate
reflux method. Suspended solids (SS) were determined by gravi-
metric method. Ammonium nitrogen (NH4 + ) and total Kjeldahl
nitrogen (TKN) were analyzed by distillation and macro-kjeldahl
methods. Table 1 shows the chemical characteristics of leachate
fed to the two-stage MBR system, respectively.
The reactor experiment focused on BPA and BHT removals in
MBR at higher concentrations of 1000 ␮g/L was conducted in order
to evaluate the biodegradation capacity of MBR sludge. These initial
concentrations were set based on their observed concentrations in
municipal landfill leachate in Thailand [6]. The concentrations were
also set to ensure their availability to assess their biodegradation
during the reactor experiment. The same concentrations were used
in batch experiments for assessing kinetic rate constant by ensur-
ing their remaining concentrations after 24 h. Previous researches
[6,16] also suggested that using high concentrations of BPA and BHT
up to 1–3 mg/L did not provide detrimental effect to the microbial
activities regarding their biodegradation capabilities of the studied
compounds. The physico-chemical properties of those compounds
are shown in Table 2.
2.2. Batch experiments for determination of biodegradation by
enriched and inhibited nitrifying sludge
The enrichment of nitrifying sludge was performed using proce-
dures described in the literature [19] with some modifications. The
MBR sludge was taken from laboratory scale MBR system during
steady state condition of long-term operation to ensure the stabil-
ity of sludge condition used in batch experiments. Nitrifying sludge
was enriched in a fill-and-draw operation with a 4-day cycle in a 2 L
reactor at room temperature for more than 4 months. At the enrich-
Table 2
Physico-chemical properties of phenolic compounds.
Compounds Chemical structure
BPA
BHT
a
The mass numbers used in the GC/MS detection in this study.
313
ment period, MBR influent leachate with ammonium was used as
an enrichment medium. The ammonium concentration was grad-
ually increased from 100 to 300 mg/L. The pH in the reactor was
controlled at 7.5–8.0 using NaHCO3 . Air pump was used to aerate
Avg. (SD)
the culture to maintain a DO level higher than 6.0 mg/L. During
28.3(0.5)
the enrichment nitrifying sludge, the NH4 + , NO2 − , NO3 − concen-
8.70(0.15)
6598(269) trations were measured. All batch experiments were performed in
9273(237) triplicate.
2476(241) Batch experiments of BHT and BPA biodegradation were per-
1250(105) formed according to the procedure described in our previous study
214(8)
119(4)
[16] and these experiments were done in parallel with the opera-
1.632(0.235) tion of the laboratory-scale MBR system. In order to differentiate
0.054(0.002) the removals of BHT and BPA by adsorption and biodegradation,
27.8(1.5) the experiments with the complete inhibition of biological activi-
ties were also carried out to determine their adsorption onto sludge.
For this purpose, the sludge inactivation was performed by pasteur-
ization at 121 ◦ C for 15 min (three times) in order to terminate all
microbial activities.
The removals by both adsorption and biodegradation were
examined using active sludge. The difference in removals between
active and inactivated sludge provide their removal through
biodegradation. Among them, the role of nitrification on BHT and
BPA degradation were also assessed by adding 20 mg/L of allyl-
thiourea (ATU) to the mixed liquor. ATU is known to inhibit the
activity of ammonia oxidizing bacteria (AOB), which is responsible
for the first step reaction in the nitrification process. By compar-
ing the biodegradation between sludge samples with and without
ATU addition, the role of nitrification in the biodegradation can be
determined.
2.3. Toxic organic micro-pollutants analyses
Solid phase extractions (SPE) were used to determination con-
centration of phenolic compounds and phthalic acid esters in
landfill leachate sample. SPE was carried out using C18-Bond Elut®
(Varian, Inc.), as SPE resin. Pre-packed columns holding 500 mg
of SPE sorbent were cleaned by pure water and followed by
solvent wash (methanol). Firstly, soluble leachate samples (typi-
cally 100 mL) were pumped through the C18-SPE columns at flow
rate of 1 mL/min. The loaded columns were dried with clean air
and then eluted by 2 × 3 mL of dichloromethane/methanol mix-
ture (1:9, v/v), respectively. The volume was reduced to 0.5 mL
by nitrogen evaporator/concentrator (ChanoVap, Amani, Thailand).
The filter residue samples were sonicated for 1 h with 50 mL of
methanol/dichloromethane mixture (1:4, v/v). The prepared sam-
ple used the same conditions and SPE column from soluble sample
extraction according to analytical organic micro-pollutants in sol-
uble sample.
Gas chromatography with mass spectrometry (GCMS-QP2010
plus, Shimadzu, Japan) was used to determine concentration of
ten toxic organic contaminants. The device was equipped with a
Cas no. log Kow SIM mass numbers (m/z)a
Target Reference ion
80–05-7 3.32 213 119
128–37-0 5.10 205 220
314 V. Boonyaroj et al. / Journal of Hazardous Materials 323 (2017) 311–318
30 m RTX-35MS capillary, 0.25 mm I.D. with thickness of 0.50 ␮m.
Helium was used as carrier gas. The quantification of BHT and
BPA in batch experiment were performed with one ␮L of sam-
ple was injected automatically (split 1 min). The chromatographic
separation process was performed by program which set col-
umn oven temperature from 150 ◦ C held for 1 min and raised to
270 ◦ C at 20 ◦ C/min held for 7 min. The ions were produced by
electron impact at 70 eV, ion source temperature at 200 ◦ C, and
interface temperature at 270 ◦ C condition. The compounds were
detected and quantified using the sim/scan mode. The organic
micro-pollutants in leachate samples were identified by com-
parison with GC/MS library (Wiley7) developed from standard
substances. The detection limits of GC/MS instrument was 1.0 ␮g/L
for phenolic compounds and phthalic acid esters measurements as
the samples were concentrated by evaporation of elution from SPE
step prior to GC/MS analysis. For checking the accuracy of analysis,
internal standard of known concentrations was used to determine
the difference between the prepared and measured concentrations
of compounds during SPE and GC/MS analysis and the differences
were found to be 9.9% and 8.7% for BHT and BPA respectively.
2.4. Determination of nitrifying bacteria in sludge
Relative percentages of nitrifying bacteria including in sludge
samples were evaluated using fluorescent in-situ hybridization
(FISH) technique at the end of experimental period. FISH analysis
protocol used in this study is described as follows.
MBR sludge and enriched nitrifying sludge were collected from
the laboratory scale MBR and the enrichment reactor, respectively.
After the samples were collected, 2 mL of mixed liquor from MBR
was taken into 50 mL polyethylene tube. The sample was cen-
trifuged at 4000 rpm during 10 min at 4 ◦ C and the supernatant
was discarded. The extraction was performed by addition 4 mL
of 8% Paraformaldehyde/PBS (8% PFA) to the sludge samples and
re-suspended the by shaking vigorously using vortex. The fixation
was then proceeded by storing the samples over night at 4 ◦ C. The
samples were then centrifuged at 4000 rpm for 12 min at 4 ◦ C and
the supernatant was discarded. The next step was the addition of
5 mL of 1x PBS and the samples were then re-suspended by vortex
(repeated washing with PBS for 3 times) followed by the addition
of 4 mL of 1x PBS and mixed with 4 mL of 99.5% ethanol. Finally, the
sample was re-suspended and stored at −20 ◦ C until hybridizing
reaction.
The samples obtained from the preparation step was car-
ried on by immobilization of 5 ␮L fixative sample onto the
gelatin coated slide and dehydration with various concentra-
tions of ethanol (50% > 80% > 95% > 50% > 80%) for 3 min for each
ethanol concentration. The oligonucleotide probes used in this
study were commercially synthesized with reverse-phase car-
tridge purification (RP1) method and fluorescently 5 labeled with
fluorescein-isothiocyanate (FITC) dye (Sigma-aldich, Singapore).
The hybridization conditions including the formamide concentra-
tion in the hybridization buffer and NaCl concentration. The probes
used in this study are shown in Table S1. For hybridization step,
6 ␮L hybridization buffer and 1 ␮L probe were overlaid on the
sample slides. The FISH assay was performed according to the
protocol described elsewhere [21] at 46 ◦ C for 2.5 h in hybridiza-
tion buffer (0.9 M NaCl, 20 mM tris-hydrochloride, 0.01% sodium
dodecyl sulfate (SDS), formamide) containing 5 ng of probe/␮L in
incubator. A negative control (no probe) was included for every
sample to observe autofluorescence. After hybrization, the slides
were rinsed and immersed in pre-warmed washing buffer (20 mM
tris-hydrochloride, 0.01% SDS, NaCl) at 48 ◦ C for 10 min. After wash-
ing, the slides were rinsed briefly with DI water, air-dried, and
mounted with a cover slip using a Prolong Gold anti-fade reagent.
They were coated ay the borders with enamel to avoid drying.
30,000
MLSS in aerobic reactor
MLVSS in aerobic reactor
25,000 MLSS in anoxic reactor
MLVSS in anoxic reactor
MLSS and MLVSS (mgL-1)
20,000
15,000
10,000
5,000
0
0 30 60 90 120 150 180 210 240 270 300
Time (days)
Fig. 2. MLSS and MLVSS concentrations in two-stage MBR.
The FISH samples were examined by fluorescence microscopy
(BX51 microscope; DP71 camera, Olympus, Japan). Barried filters,
UMW-B2, UMW-G2 and UMW-U2 were used to collect the excited
fluorescence of the FITC-labeled probes, and the Rhodamine Red-
labeled probes for target organisms [21,22] and DAPI blue-labeled
probes respectively. The cellSens dimension imaging software ver-
sion 1.4.1 was used as an image analysis tool to help in determining
the relative area taken up by target cells complimentary to the
specific probe compared to the area of cells complimentary to the
EUB338 probe. The average area fraction was determined by eval-
uating at least nine representative microscopic fields.
The quantification of ammonia oxidizing bacteria and nitrite
oxidizing bacteria of sludge from laboratory-scale two-stage MBR
system, and sludge used in batch experiment were determined
by FISH technique to find out the information supported about
the microorganisms responsible in micro-pollutant removals. The
percentage of Ammonia oxidizing ˇ Proteobacteria, and Nitrobacter
spp. in anoxic and MBR sludge were determined from the relative
percentage of NSO1225, and NIT3 probe, respectively. The quan-
tification of EUB338 probe was determined by specification for all
bacteria. The percentages were determined by evaluating at least
nine representative microscopic fields.
3. Results and discussion
3.1. Treatment performance of two-stage MBR
During the laboratory-scale two-stage MBR operation, aver-
age BOD and COD concentrations in landfill leachate were 6598
and 9273 mg/L yielding BOD and COD loading rates of 6.6 and
9.3 kg/m3 d, respectively. Average TKN loading rates were between
0.20-0.23 kg/m3 d during the operation period. The performance of
laboratory scale MBR in terms of organic (BOD, COD) and nitro-
gen (NH4 + , TKN) removals are presented in Table 3. Corresponding
biomass variations in laboratory scale two-stage MBR system are
shown in Fig. 2. During the long term operation, the aerobic reac-
tor removed 99% of BOD and 96% of COD on average. NH4 + and TKN
removals were 90% and 85%, respectively. The treatment perfor-
mance was found to be relatively stable over the entire operation
period despite gradual change in sludge biomass. MLSS concentra-
tion in aerobic reactor was gradually increased from 5.6 to 10.7 g/L.
MLVSS/MLSS ratio of biomass was kept between 0.48-0.58. Most of
the removals were taken place in the aerobic reactor as less than
20% removal was observed in the anoxic reactor.
The biomass concentration in anoxic reactor was raised to 27 g/L
during 300 days of operation whereas the biomass concentration
in aerobic reactor was kept constantly at about 11 g/L by the
 V. Boonyaroj et al. / Journal of Hazardous Materials 323 (2017) 311–318 315

Table 3
Treatment performance of two-stage MBR.

Parameter Effluent concentration (mgL−1 )

Anoxic reactor Aerobic reactor Total removal efficiencies (%)

Range Avg. (SD) Range Avg. (SD) Range Avg.(SD)

BOD 5120–6975 6115(492) 60–112 67(18) 96–100 99(3)

COD 6125–8850 7990(328) 340–465 385(40) 87–98 96(2)
TOC 2050–2630 2520(205) 218–240 230(8) 85–94 91(7)
NH4 + 114–135 129(10) 3–26 12(5) 88–98 90(5)
TKN 200–240 230(11) 32–58 33(12) 72–90 85(3)

re-circulation operation. An introduction of sludge re-circulation
and increasing sludge age could affect the biofloc size and EPS pro-
duction in the sludge. The determination of particle size of biofloc
did not suggest any significant change in sludge particle size in the
aerobic reactor with time. Majority of them lied within the range
of 10–100 ␮m. EPS in term of protein was found higher than car-
bohydrate and they were found as bound EPS more than soluble
form. Protein content of soluble EPS and bound EPS were found
39 and 248 mg/g dried solids, respectively. The ratio of protein
to carbohydrate (P/C) of bound EPS and soluble EPS were found
to be 23.16, and 37.20, respectively. The EPS compositions, such
as protein and carbohydrate, strongly influence the surface prop-
erties such as hydrophobicity and surface charge of sludge, and
hydrophobic fraction of bound EPS was made up only proteins.
Moreover, hydrophobicity of sludge also influent its capacity to
remove micro-pollutants through surface adsorption.
3.1.1. Removals of toxic organic micro-pollutants in MBR
It was found that the removal efficiencies of BPA and BHT had
an increasing trend along the operation period. Between them,
the removal efficiencies of BHT were higher than BPA. Despite
the biomass variations in the system, the removal efficiencies
of the studied compounds were not adversely affected at the
concentration of 1000 ␮g/L each compound added into the reac-
tor. The removal efficiencies of those organic micro-pollutants in
laboratory-scale MBR were summarized in Table 4. The results
show that anoxic reactor could remove BPA and BHT by only 3–4%,
and their removals were taken place mainly in the aerobic reac-
tor. The removal efficiencies of those compounds were 65% for
BPA and 70% for BHT. The octanol-water partition coefficient (Kow )
of compounds were found affected their removals during reactor
operation. BHT with log Kow of 5.10 was found removed higher
than BPA with log Kow of 3.32. Previous study [23] reported that
the micro-pollutant removals were probably benefit from com-
plete retention of solids in MBR. On the other hand, high SRT
were needed to achieve high sludge concentrations, typical for
MBRs. The removals of BPA observed in this study was found
comparable lower than those reported for nitrifying immobilized
biomass (87.1–92.9%) operated at high loading of 7.0 kgCOD/m3 .d
and 1.0 kgN/m3 .d with BPA feeding concentrations of 2.5–10 mg/L
[24]. In the mentioned study, to high organic loading employed
promoted the removals of BPA mainly by heterotrophic bacteria.
Meanwhile, it was also reported that the contribution of het-
erotrophs on BPA removal could be limited under low organic
but high ammonium loading during which BPA removal rate was
found much slower (60–70% after 24 h) [18]. For several polar pol-
lutants, the degradation could not directly relate to a change in
operational parameters such as sludge concentration, sludge load,
organic load, and other operational parameter due to the require-
ment for microbial adaptation. MBR treatment seems to enhance
removal of micro-pollutants with intermediate biodegradability.
For easily degradable and intrinsically recalcitrant compounds,
MBRs could not make a significant difference in terms of overall
removal efficiencies. It suggests that MBR treatment turned out to
be less sensitive to operational variables such as HRT and will hence
show a higher robustness than conventional systems especially in
term of micro-pollutant removal through microbial biodegrada-
tion.
3.2. Biodegradation of BPA and BHT
In order to investigate the role of nitrifying organisms in
the sludge on the biodegradation of BPA and BHT, batch exper-
iment using non-enriched, and enriched nitrifying sludge, and
sludge with inhibitors were performed. The MBR sludge was taken
from laboratory scale reactor. Nitrifying sludge was enriched for
more than 4 months. At the enrichment period, MBR influent
leachate with ammonium was used as enrichment medium. The
ammonium concentration was gradually increased from 100 to
300 mg/L. The nitrification process is performed by a group of
autotrophic microorganisms. The principal mechanism for the
nitrogen removal takes place by two reactions, one is ammonia
oxidized to nitrite by Nitrosomonas spp. and the other is nitrite
to nitrate by Nitrobacter spp. The ammonium concentration was
consumed during the enrichment nitrifying bacteria, and transfor-
mation to nitrate concentration as shown in Fig. 3.
During the 24 h of enrichment nitrifying sludge, ammonium
was oxidized by Nitrosomonas spp. and nitrate was produced by
Nitrobacter spp. The large difference in NH4 + reduction (250 mg/L),
and nitrate production during batch experiment was mainly due
to N uptake for heterotrophic growth as the influent leachate con-
tained initial BOD concentration of 5000 mg/L.
The enrichment of nitrifier culture is a significant factor on
the biodegradation of XOCs in biological wastewater treatment
system [25,26]. It is therefore possible that nitrifying organisms
was also responsible for the removal of micro-pollutants through
their co-metabolic pathways [27]. The MBR sludge collected from
laboratory-scale two-stage MBR system was used in batch experi-
ment The enrichment of nitrifying sludge was performed by adding
NH4 + concentrations of 100–300 mg/L as substrate. In the compa-

Table 4
Phenolic compound removals in two-stage MBR.

Compound Initial concentration (␮gL−1 ) Concentration (␮gL−1 ) Removal efficiency (%)

Effanoxic In MBR Eff aerobic %Ranoxic %R aerobic

BPA 1000 965(20)* 615(31)* 350(14)* 3 65

BHT 1000 952(25)* 652(16)* 300(8)* 4 70
*
Average (SD) values, No. of samples = 6.
316 V. Boonyaroj et al. / Journal of Hazardous Materials 323 (2017) 311–318

350 1,000
Enriched sludge
900
300
NH4-N concentration (mgL-1)
Non-enriched sludge

BPA concentration (µgL-1)

800
250
700
200 600
500
150
400
100
300
50 200
100
0
0 12 24 36 48 60 72 84 96 0
Time (h) 0 3 6 9 12 15 18 21 24

(a) Time (h)

50 (a)
Enriched sludge 1,000
45
Non-enriched sludge
NO3-N concentration (mgL-1)

900
40

BHT concentration (µgL-1)

35 800

30 700

25 600
20 500
15 400
10 300
5 200
0 100
0 12 24 36 48 60 72 84 96
0
Time (h)
0 3 6 9 12 15 18 21 24
(b)
Time (h)
Biotic sludge with nitrifying activity
Fig. 3. NH4 -N (a), and NO3 -N (b) concentration profiles in batch experiments using
Biotic sludge w/o nitrifying activity
enriched and inhibited nitrifying sludge. Abiotic sludge with ammonia
Abiotic sludge w/o ammonia
Biotic sludge with ATU inhibitor
rability with all bacteria, enriched nitrifying sludge that used in
(b)
batch experiment found that the percentage of Ammonia oxidizing
ˇ Proteobacteria, and Nitrobacter spp. were higher than non-enrich Fig. 4. BHT (a) and BPA (b) removals by enriched and inhibited nitrifying sludge.
nitrifying sludge in the percentage of 68.5%, and 23.1%, respectively.
The results reveal the similarity between the Ammonia oxidizing ˇ
Proteobacteria, and Nitrobacter spp. in both reactors. the concentrations of BHT and BPA were reduced by 120 and
Comparatively, the higher percentage of MBR sludge, hybridized 125 ␮g/L, respectively for enriched nitrifying sludge with NH4 +
with NSO1225, specific for the detection of Ammonia oxidizing ˇ addition. For enriched nitrifying sludge without NH4 + addition,
Proteobacteria, and NIT3 with specific for the detection of Nitrobac- the concentrations of BHT and BPA were reduced by 160 and
ter spp. were found in the aerobic reactor. Table 5 presents the 280 ␮g/L. The concentrations of BHT and BPA were reduced by 150
relative percentage of Ammonia oxidizing ˇ Proteobacteria, and and 200 ␮g/L, respectively of enriched nitrifying sludge with ATU.
Nitrobacter spp. to the total microorganisms in sludge collected Table 6 shows the removal rate, and of these compounds at 24 h
from laboratory-scale MBR, and enriched nitrifying sludge that of batch experiment. These results confirm the observation of BHT
used in batch experiment. and BPA removals in laboratory-scale MBR experiment suggesting
Non-enrich, and enriched nitrifying sludge activity testing were higher removal efficiencies of BHT as compared to BPA. Neverthe-
performed to investigate the biodegradability of two selected less, their higher removals was resulted from better adsorption
phenolic compounds. The concentrations of MLSS in these batch onto sludge due to its hydrophobic property.
experiments were controlled at 1000 mg/L. The initial concentra- The total removal (adsorption plus biodegradation) rates of
tions of BHT, and BPA were set at 1000 ␮g/L. The concentration of BHT by enriched nitrifying sludge with NH4 + addition, enriched
BPA, and BHT removal in batch experiments using non-enrich, and nitrifying sludge without NH4 + addition, and enriched nitrifying
enriched nitrifying sludge were decreasing rapidly via biodegrada- with ATU were 0.1078 h−1 , 0.0967 h−1 , and 0.0974 h−1 , respectively
tion mechanisms as shown in Fig. 4. During 24 h of biodegradation, (Fig. 5). Meanwhile, the total removal rates of BPA were 0.0958 h−1 ,

Table 5
Relative percentage of Ammonia oxidizing ˇ Proteobacteria, and Nitrobacter spp. to the total microorganisms in sludge.

Bacterial group (probe) Laboratory-scale Batch experiment

Anoxic sludge MBR sludge Enriched nitrifying sludge

Ammonia oxidizing ˇ Proteobacteria (NSO1225) 3.98a (26.2b ) 14.54a (22.3b ) 21.80a (25.9b )
Nitrobactor spp. (NIT3) 3.14a (9.1b ) 5.03a (9.5b ) 8.90a (14.2b )
a
% of DAPI.
b
% among all bacteria.
 V. Boonyaroj et al. / Journal of Hazardous Materials 323 (2017) 311–318 317

Table 6
Removals of BPA and BHT by enriched nitrifying sludge with and without inhibitors.

Enriched nitrifying sludge Removal rates (h−1 ) at 24 h Removal efficiency (%)

Total removal Adsorption Adsorption Biodegradation

BHT BPA BHT BPA BHT BPA BHT BPA

without-NH4 + 0.0967 0.0664 0.0261 0.0119 27 18 73 82

with ATU inhibitor 0.0974 0.0756 0.0265 0.0111 27 15 73 85
with NH4 + 0.1078 0.0958 0.0266 0.0119 25 12 75 88

0 0

ln C/Co of BPA (Total removal)

y = -0.0967x + 0.4479 y = -0.0664x + 0.3192
ln C/Co of BHT (Total removal)

R² = 0.9728 (without ammonia) R² = 0.9737 (w/o ammonia)

-0.5 -0.5

-1
-1
y = -0.0974x + 0.4097 y = -0.0756x + 0.3377
R² = 0.9791 (with ATU) R² = 0.9698 (with ATU)
-1.5
-1.5

-2
-2 y = -0.0958x + 0.3116
y = -0.1078x + 0.4118 R² = 0.9858 (with ammonia)
R² = 0.9808 (w/o ammonia) -2.5
-2.5 0 3 6 9 12 15 18 21 24
0 3 6 9 12 15 18 21 24 Time (h)
Time (h) Biotic sludge with ammonia
Biotic sludge w/o ammonia
Biotic sludge with ammonia Biotic sludge with ATU inhibitor
Biotic sludge w/o ammonia
Biotic sludge with ATU inhibitor (a)
(a) 0
0
y = -0.0261x + 0.1226 -0.05 y = -0.0119x + 0.0224
ln C/Co of BPA (Adsorption)

R² = 0.912 (w/o ammonia)

ln C/Co of BHT (Adsorption)

-0.1 R² = 0.9393 (w/o ammonia)

-0.1
-0.2
y = -0.0265x + 0.1243
R² = 0.9393 (with ATU) -0.15
-0.3 y = -0.0111x + 0.0092
R² = 0.8731 (with ATU)
-0.4 -0.2

-0.5 y = -0.0266x + 0.1193 -0.25

y = -0.0119x + 0.0112
R² = 0.938 (with ammonia)
R² = 0.8991 (with ammonia)
-0.6 -0.3
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time (h) Time (h)
Abiotic sludge with ammonia
Abiotic sludge with ammonia
Abiotic sludge w/o ammonia
Abiotic sludge with ATU inhibitor Abiotic sludge w/o ammonia
Abiotic sludge with ATU inhibitor
(b)
(b)
Fig. 5. Total removal rate of BHT (a) and adsorption rate of BHT (b) using enriched
and inhibited nitrifying sludge. Fig. 6. Total removal rate of BPA (a) and adsorption rate of BPA (b) using enriched
and inhibited nitrifying sludge.

0.0756 h−1 , and 0.0664 h−1 , respectively (Fig. 6). The removal effi-
ciencies of BHT through biodegradation mechanism by enriched biodegradation rate obtained for BHT and BPA removals by MBR
nitrifying sludge with NH4 + addition, enriched nitrifying sludge sludge obtained from this study was 0.0812 and 0.0839 h-1 com-
without NH4 + addition, and enriched nitrifying with ATU were parable to 0.079 and 0.087 h−1 observed for MBR sludge with
more than 73%, respectively. Meanwhile, the removal efficiencies nitrification activity even though heterotrophic nitrifiers could be
of BPA through biodegradation mechanism by enriched nitrifying the main microbial group responsible for the biodegradation of
sludge with NH4 + addition, enriched nitrifying sludge without NH4 + micro-pollutants in that study.
addition, and enriched nitrifying with ATU were 88%, 82%, and 85%, The removal rates through their co-metabolic pathways on BHT
respectively. The presence of nitrification improved the biodegra- and BPA removal were found to be 0.0106 h−1 , and 0.0294 h−1 ,
dation rate of BPA to a greater extent (44%) as compared to those of respectively. By comparing the results between those experiments,
BHT (11%). This observation confirmed the role of co-metabolism it was found that the presence of nitrifying activities in enriched
provided through nitrification reaction on the enhancement of sludge could improve BPA and BHT biodegradation by 7% and
micro-pollutant biodegradation especially for hydrophilic com- kinetic rate by 44%. The results also suggested that, not only nitri-
pounds. In comparison to our previous investigation [28], the fying bacteria but heterotrophic organisms also help degrading
318 V. Boonyaroj et al. / Journal of Hazardous Materials 323 (2017) 311–318
significant amount of phenolic compounds in MBR operated under
long sludge age condition. Nevertheless, high nitrifying activities
of microorganisms were found associated with enhanced removal
of BHT and BPA biodegradation. Recent studies [26,29,30] have
also demonstrated the role of nitrifying organisms and nitrifica-
tion condition on the removals of micro-pollutants in other types
of wastewater where the finding from this study has confirmed
its significance on the removals of toxic organic compounds in
nitrogen-rich landfill leachate.
4. Conclusions
This research provides a perspective on application of culti-
vated and enriched nitrifying MBR sludge operated without sludge
wastage to remove phenolic compounds presented in landfill
leachate. The removals of BPA and BHT in MBR were 65% and 70%
at influent concentrations of 1000 ␮g/L in which the biodegrada-
tion were mainly taken place in the aerobic reactor. The enriched
nitrifying sludge could enhance the biodegradation rate of BPA
and BHT compared to non-enriched sludge and their removal rates
through co-metabolic pathways were found to be 0.0106 h−1 , and
0.0294 h−1 . The presence of nitrifying activities in enriched sludge
could improve BPA and BHT biodegradation by 7% and kinetic rate
by 44%. This study confirmed the role of co-metabolism provided
through nitrification reaction on the enhancement of biodegrada-
tion of toxic organic micro-pollutants presented in landfill leachate.
Novelty statement
The research has identified the role of nitrifying microorganisms
in enhancing biodegradation of phenolic compounds presented in
municipal solid waste leachate. During long term operation of two-
stage membrane bioreactor (MBR) under no excess sludge wastage
condition, heterotrophic and autotrophic microbial communities
capable for biodegradation of complexed toxic organic compounds
were developed. Their ability to biodegrade high concentrations
of BPA and BHT (up to 1 mg/L) under the presence and absence
of nitrifying activities were evaluated. The results revealed the
enhancement of the biodegradation degree by 7% and the kinetic
rate by 44% under the presence of nitrifying activities.
Acknowledgement
The authors would like to express their appreciation to Japan
International Cooperation Agency (JICA) for providing research
fund through Research and Development for Water Reuse Tech-
nology in Tropical Regions (Water-Intro) project.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jhazmat.2016.06.
064.
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