Accepted Manuscript

Use of aged sludge bioaugmentation in two-stage activated sludge system to

enhance the biodegradation of toxic organic compounds in high strength wastewater

Jarungwit Boonnorat, Somkiet Techkarnjanaruk, Ryo Honda, Sivakorn Angthong,

Nimaradee Boonapatcharoen, Sutharat Muenmee, Pradthana Prachanurak

PII: S0045-6535(18)30501-0
DOI: 10.1016/j.chemosphere.2018.03.084
Reference: CHEM 21028

To appear in: ECSN

Received Date: 3 September 2017

Revised Date: 12 March 2018
Accepted Date: 12 March 2018

Please cite this article as: Boonnorat, J., Techkarnjanaruk, S., Honda, R., Angthong, S.,
Boonapatcharoen, N., Muenmee, S., Prachanurak, P., Use of aged sludge bioaugmentation in two-stage
activated sludge system to enhance the biodegradation of toxic organic compounds in high strength
wastewater, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.03.084.

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 ACCEPTED MANUSCRIPT

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1 Use of aged sludge bioaugmentation in two-stage activated sludge system
2 to enhance the biodegradation of toxic organic compounds in high
3 strength wastewater
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5 Jarungwit Boonnorat a,b,*, Somkiet Techkarnjanaruk c,d, Ryo Honda e, Sivakorn Angthong f,
6 Nimaradee Boonapatcharoen c, Sutharat Muenmee g, Pradthana Prachanurak h

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a
8 Environmental Engineering Program, Faculty of Engineering, Rajamangala University of

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9 Technology Thanyaburi (RMUTT), Klong 6, Pathum Thani 12110, Thailand
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b
11 Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology
12 Thanyaburi (RMUTT), Klong 6, Pathum Thani 12110, Thailand
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c
14 Excellent Center of Waste Utilization and Management (ECoWaste), King Mongkut’s University
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15 of Technology Thonburi (KMUTT), Bangkhuntien, Bangkok 10150, Thailand
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17 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and
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18 Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand

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20 Faculty of Environmental Design, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
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21 Japan
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23 Department of Industrial Engineering, Faculty of Engineering, Rajamangala University of
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24 Technology Thanyaburi (RMUTT), Klong 6, Pathum Thani 12110, Thailand

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26 Faculty of Science, Energy and Environment (SciEE), King Mongkut’s University of Technology
27 North Bangkok (Rayong Campus), Rayong 21120, Thailand
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29 Department of Civil and Environmental Engineering, Faculty of Engineering, Srinakharinwirot
30 University, Ongkharak, Nakhon Nayok 26120, Thailand
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33 * Corresponding author: Jarungwit Boonnorat, D.Eng.
34 Tel. +662-549-4180 (Fax) +662-549-4119
35 Email: jarungwit_b@rmutt.ac.th , jarungwit.b@gmail.com
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2 Abstract
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4 This research investigates the toxic organic compounds biodegradation efficiency of two-stage
5 activated sludge systems with (bioaugmented) and without aged sludge bioaugmentation (non-
6 bioaugmented). The influent was a mixture of leachate and agriculture wastewater (1:1, v/v), used as
7 the representative high strength wastewater. The bioaugmented and non-bioaugmented systems were

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8 operated in parallel, with three levels (low, moderate, and high) of concentrations of organics,
9 nitrogen, and toxic organic compounds in the influent (conditions 1, 2, and 3). The results showed

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10 that both systems could efficiently degrade the organic compounds. Nevertheless, the toxic organic
11 compounds biodegradation efficiency of the bioaugmented system was higher than that of the non-

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12 bioaugmented one. The bioaugmentation enhanced the overall removal efficiency under conditions 1
13 and 2. However, the bioaugmented system became less effective under condition 3. Further analysis
14 indicated that the bacterial groups essential to the toxic organic compounds biodegradation were

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15 abundant in the aged sludge, including heterotrophic bacteria, heterotrophic nitrifying bacteria, and
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16 nitrifying bacteria. The abundance of the effective bacteria improved the biodegradation and
17 wastewater treatment performance of the bioaugmented system. In essence, the aged sludge
18 bioaugmentation is a viable and eco-friendly solution to improving the treatment efficiency of the
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19 biological activated sludge system, despite limited biodegradation efficiency in an elevated

20 compounds-concentration environment.
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23 Keywords: Bioaugmentation ; Aged sludge ; Micropollutants ; Wastewater treatment
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2 1. Introduction
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4 Toxic organic compounds in water resources, including endocrine disrupting compounds (EDCs),
5 pharmaceuticals, and personal care products, are harmful to aquatic ecosystems and human health
6 (Tran et al., 2013a; Arriaga et al., 2016). Many toxic organic compounds are degradation-resistant

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7 and are only partially degraded in a conventional wastewater treatment system. However, most
8 developing countries, including Thailand, adopt the biological activated sludge (AS) wastewater
9 treatment technology due to the low financial investment, ease of operation, and environmental

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10 friendliness, despite the limited toxic compounds degradation efficiency of the technology.
11

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12 Leachate is a type of high strength wastewater containing organic compounds, nitrogen, and toxic
13 compounds in elevated concentrations. The compounds present in leachate include certain
14 pharmaceuticals, phenolic compounds, and phthalic acid esters (PAE), which are the chemical
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compounds found in plastics. The World Health Organization and the US Environmental Protection
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16 Agency classify the phenolic compounds and PAE (i.e., bisphenol A (BPA), 2,6-di-tert-butyl-phenol
17 (2,6-DTBP), di-butyl-phthalate (DBP), and di-(ethylhexyl)-phthalate (DEHP)), as the EDCs that
18 disrupt the neural development and hormonal systems, as well as contribute to the increased
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19 incidence of cancers (Huang et al., 2010).

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21 Boonyaroj et al. (2017) enriched a membrane bioreactor (MBR) with nitrifying sludge and found
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22 that the nitrifying sludge promoted the biodegradation of toxic organic compounds in landfill
23 leachate. Park et al. (2017) studied the removal of pharmaceuticals and personal care products in an
24 MBR by ammonia oxidizing bacteria (AOB) and found that AOB played a key role in removing
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25 these compounds in the MBR system.

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27 Bui et al. (2016) noted that, due to the degradation-resistant nature of many toxic compounds, the
28 removal of toxic compounds is more effective by using advanced treatment technologies, e.g., the
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29 advanced oxidation process (AOP), or the MBR. However, the aim of this research is to explore
30 ways to enhance the biodegradation efficiency of the economical and environmentally-friendly AS
31 technology.
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33 Bioaugmentation is a remedial strategy to enhance wastewater treatment efficiency by introducing
34 microorganisms into the wastewater reactor to catalyze the catabolism of refractory organic
35 compounds (Herrero and Stuckey, 2015). The technique promotes the bacterial abundance and
36 diversity, which enhances the nitrogen removal and wastewater treatment efficiency (Chen et al.,
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1 2015). Examples of bioaugmentation include the use of a single modified bacterial strain (Qu et al.,
2 2009; Yu et al., 2010), modified bacterial consortia (Guo et al., 2010), and biodegradation-relevant
3 genes (e.g., aged sludge).
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5 Guo et al. (2010) augmented a wastewater treatment system with Proteobacteria, Bacterioides,
6 Nitrospirales, Cyanobacteria, and Bacillus. The resulting effluent contained less than 660 mg L-1 of
7 COD and 8 mg L-1 of NH3-N. Qu et al. (2009) studied the removal of bromoamine acid (dye) in a

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8 synthetic wastewater by using a lab-scale MBR bioaugmented with Sphingomonas xenophaga QYY.
9 The bioaugmented system could achieve the COD and bromoamine acid removal efficiencies of 30-

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10 50% and 80-90%, respectively. Yu et al. (2010) assessed the removal of o-Nitrobenzaldehyde
11 (ONBA) in the synthetic wastewater augmented with Pseudomonas putida ONBA-17gfp by using a

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12 lab-scale sequencing batch reactor (SBR). They reported the complete removal of the compound and
13 the COD removal of 96%.
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15 Existing research on enhanced toxic organic compounds biodegradation has focused on advanced
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16 chemical and/or biological treatment technologies that require costly bioaugmenters. On the other
17 hand, there exists no study on the enhanced biodegradation efficiency of toxic organic compounds
18 by using the conventional biological AS system augmented with aged sludge.
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20 According to Boonnorat et al. (2014a, 2017), aged sludge is the microbial sludge acclimatized more
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21 than a year in a high strength wastewater treatment system. The bacterial communities in aged
22 sludge are more diverse and more abundant than seed sludge (non-acclimatized), especially the
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23 bacterial groups essential for the biodegradation of toxic organic compounds, including
24 heterotrophic bacteria (HB), heterotrophic nitrifying bacteria (HNB), ammonia oxidizing bacteria
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25 (AOB), and nitrite oxidizing bacteria (NOB).

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27 Thus, this research investigates the biodegradation of toxic organic compounds in high strength
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28 wastewater of two-stage AS schemes (consisting of the anoxic and aerobic tanks) with
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29 (bioaugmented system) and without aged sludge bioaugmentation (non-bioaugmented system). Both
30 systems were operated in parallel, with three levels of concentrations of organics, nitrogen, and toxic
31 organic compounds in the influent: low (condition 1; days 0-77), moderate (condition 2; days 78-
32 161), and high concentration levels (condition 3; days 162-245). The high strength wastewater was a
33 mixture of leachate and agriculture wastewater (1:1, v/v). The target toxic organic compounds were
34 BPA, 2,6-DTBP, DBP, DEHP, carbamazepine (CBZ), diclofenac (DCF), and N, N-Diethyl-meta-
35 toluamide (DEET).
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37 2. Materials and methods
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2 2.1. Optimal aged sludge bioaugmentation ratio and kinetics of toxic organic compounds
3 biodegradation in batch experiment
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5 The optimal aged sludge bioaugmentation ratio for effective biodegradation of the target toxic
6 organic compounds was determined in batch experiments by using 2-liter flasks. First, the
7 adsorption of the toxic compounds was observed for 24 h, and the adsorption equilibrium was

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8 determined by inhibition of microbial sludge activity by NaN3 (Barbot et al., 2010). The equilibrium
9 concentrations were incorporated into the initial concentration (1 mg L-1) for the subsequent

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10 biodegradation analysis. Supplementary Figure 1 shows the adsorption equilibriums of the target
11 toxic organic compounds. The biodegradation performance was examined under the aerobic

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12 condition because the target toxic organic compounds were effectively degradable in this
13 environment (Boonnorat et al., 2014b). The batch-experiment biodegradation was observed for 24 h,
14 which is the hydraulic retention time (HRT) of the system operation.

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16 The aged sludge was from our previous study, which had been cultivated in high strength
17 wastewater longer than a year (Boonnorat et al., 2017). The mixed liquor suspended solids (MLSS)
18 of the aged sludge was 2 g L-1, and that of the seed sludge was 7 g L-1. The aged sludge
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19 bioaugmentation ratio was varied between 10%, 20%, and 30% (v/v), given the MLSS concentration
20 of 7 g L-1.
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This research experimented with the relatively low bioaugmentation ratios (10-30%, v/v) in order to
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23 maintain the MLSS in the anoxic and aerobic tanks at 7 g L-1. Our preliminary experiments showed
24 that a ratio beyond 30% resulted in the MLSS exceeding 7 g L-1. The 20% bioaugmentation ratio
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25 was selected because it could efficiently degrade the target toxic organic compounds, relative to at
26 the 10% ratio. Meanwhile, at 30%, the biodegradation performance increased at a decreasing rate.
27 All the batch-scale experiments were carried out in triplicate, including the seed sludge, 10%, 20%,
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28 and 30% (v/v) aged sludge bioaugmentation.

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30 In this research, the biodegradation rates of toxic organic compounds followed a first-order kinetic
31 expression. The first-order biodegradation rate constants (k) were derived from the slope of the
32 linear plot between ln (C/C0) and t, where C0 and C are the initial concentration of the target toxic
33 organic compounds in aqueous solution and that at time t (Boonyaroj et al., 2017).
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35 2.2. Two-stage bioaugmented and non-bioaugmented AS systems
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1 Two 20-liter acrylic tanks, one containing the anoxic mixed liquor and the other the aerobic mixed
2 liquor, were used in the bioaugmented and non-bioaugmented systems (i.e., two tanks each for the
3 bioaugmented and non-bioaugmented systems). Each tank had a working volume of 10 liters. The
4 HRT of both systems was 24 h, which is the optimal HRT for the biodegradation of toxic organic
5 compounds (Boonnorat et al., 2014b). During the operation, the sludge in the sedimentation tank
6 was recirculated to the anoxic tank on a daily basis to maintain the anoxic condition and MLSS in
7 the system. The solid retention time (SRT) of the seed sludge of both systems was 30 d, which is the

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8 normal practice of local AS treatment plants.
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10 The seed sludge was obtained from a local AS wastewater treatment plant in Thailand’s capital
11 Bangkok (7 g L-1 MLSS). The influent was a mixture of leachate and agriculture wastewater and

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12 used as the representative high strength wastewater contaminated with toxic organic compounds.
13 The leachate was obtained from a municipal solid waste landfill and the garbage trucks operating on
14 the university premises (RMUTT), and the agriculture wastewater was from the farmlands around

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15 the university, which contained high concentrations of nitrogen and DEET (Boonnorat et al., 2017).
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16 Supplementary Table 1 tabulates the characteristics of the leachate and agriculture wastewater.
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18 The bioaugmented and non-bioaugmented systems were operated in parallel, with three levels of
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19 concentrations of organics, nitrogen, and toxic organic compounds in the influent: the low (condition
20 1; days 0-77), moderate (condition 2; days 78-161), and high concentration levels (condition 3; days
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21 162-245). Table 1 presents the characteristics of the three experimental influents.

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23 Insert Table 1
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25 2.3. Water quality and toxic organic compounds analysis

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27 The samples of the influent, anoxic and aerobic mixed liquors, and effluent were assessed weekly
28 according to the standard analytical procedures in the Standard Methods for the Examination of
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29 Water and Wastewater (APHA, 2005). The toxic organic compounds were extracted by solid phase
30 extraction (SPE), and the concentrations were determined by gas chromatography and mass
31 spectrometry (GC-MS) (GCMS-QP2010, Shimadzu, Japan) and liquid chromatography and mass
32 spectrometry (LC-MS) (Shimadzu, Japan) (Tadkaew et al., 2011; Tran et al., 2013).
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34 In GC-MS analysis, VertiPakTM-C18 tubes (Vertical Chromatography, Thailand) were used for
35 extracting BPA, 2,6-DTBP, DBP, and DEHP. The tubes were cleaned with 10 mL methanol
36 (MeOH) (HPLC grade, Fisher, USA) and then with 10 mL distilled water and left to dry. The tubes
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1 were filled with 300 mL water samples each and eluted with 10 mL MeOH. The eluted fractions
2 were concentrated by using a pressure blowing concentrator (Chrom Tech-CT, Taiwan) in pure
3 nitrogen gas before the GC-MS analysis. The analysis of BPA, 2,6-DTBP, DBP, and DEHP
4 followed Boonnorat et al. (2014b).
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6 In LC-MS analysis, the tubes (Oasis HLB cartridges (Waters), 250 mg bed) were cleaned with 10
7 mL MeOH and then with 10 mL distilled water and left to dry. The tubes were each filled with 300

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8 mL water samples and eluted with 10 mL MeOH. The eluted fractions were concentrated by using
9 the pressure blowing concentrator (Chrom Tech-CT, Taiwan) in nitrogen gas before the LC-MS

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10 analysis (Tran et al., 2013b). CBZ, DCF, and DEET were analyzed according to Tadkaew et al.
11 (2011). For the adsorption, 300 mL sludge samples were shaken in 10 mL MeOH for 30 min and

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12 transferred to the SPE tubes for GC-MS and LC-MS analysis.
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14 2.4. Bacterial community analysis

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16 2.4.1. Quantitative real-time polymerase chain reaction (qPCR)
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18 In the qPCR analysis, the sludge samples included the seed sludge (day 0), the aged sludge, the
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19 anoxic and aerobic mixed liquors (days 77, 161, and 245). The bacterial DNA were extracted by a
20 ChargeSwitch® gDNA Mini Bacteria Kit (Invitrogen, USA) and cleaned up by a PureLink®
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21 Genomic DNA purification kit (Invitrogen, USA). The qPCR was used to determine the DNA
22 numbers of the bacterial communities and to classify the target bacterial groups in the sludge
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23 samples. Total bacteria were quantified by 338F/518R and the AMO-producing amoA gene by
24 Amo1F/Amo2R. AOB were quantified by CTO1894/8f, CTO189Cf, and CTO654r. HNB were the
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25 difference between amoA gene and AOB. The genera Nitrobacter and Nitrospira were quantified by
26 P338f/NIT3 and NSR1113F/NSR1264R, respectively. The DNA number of the nitrifying bacteria
27 was determined from AOB and NOB. Supplementary Table 2 summarizes the primers used in this
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28 research. The standard DNA of each bacterial group was prepared in the range of 1 x 103 – 1 x 1015
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29 copies. The qPCR analysis was carried out in duplicate for each extracted DNA by using Brilliant II
30 SYBR Green QPCR Master Mix (Stratagene, USA) (Limpiyakorn et al., (2005), (2011); Zeng et al.
31 (2014); Khemkhao et al. (2016)).
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33 2.4.2. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)
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35 The bacterial DNA were amplified by the PCR technique using the primer set EUB8F/U1492R
36 (Invitrogen, USA) in the first round to increase the PCR sensitivity and then with the specific primer
37 set 338GC-F/518R (Invitrogen, USA) to identify the dominant bacterial communities. The PCR
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1 amplification condition was as follows: 50 µL PCR reaction containing 1 µL of 20 ng µL-1 DNA
2 template, 1x PCR buffer, 3 mM MgCl2, 200 µM deoxynucleoside triphosphates (dNTPs), 10 pmol of
3 each primer, and 1 U of Taq DNA polymerase (Qiagen, Germany). The PCR amplification was
4 conducted in a T100TM Thermal Cycler (BioRad, USA). The thermal program was as follows: the
5 initial denaturation at 95 oC for 5 min, followed by 30 cycles of denaturation at 95 oC for 50 s,
6 annealing at 55 oC for 30 s, and extension at 72 oC for 40 s. Afterward, the DNA were extended at 72
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7 C for another 7 min. The size and amount of the PCR products were visualized in 1.2% (w/v)

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8 agarose gel (Invitrogen, USA), and the DNA quantity was determined by a NanoDrop 1000
9 spectrophotometer (Thermo Scientific, USA).

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11 The PCR products were analyzed by a DGGE-2000 system apparatus (CBS Scientific Company,

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12 USA). In the analysis, the PCR products were amplified in 8% polyacrylamide gel (denaturant) with
13 40-60% gradient. Approximately 80% of the denaturant contained 6 M urea and 40% (v/v)
14 formamide in 1x TAE and the remaining 20% was without 6 M urea. The DGGE was carried out at

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15 80 V and 60 oC for 16 h. The denaturant was stained by SYBR Gold nucleic acid gel (Invitrogen,
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16 USA) for 15 min before visualizing on a UV transilluminator, and the images were captured by
17 Biovision CN 1000/26M (VilberLourmat, France).
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19 The DGGE profiles were generated from the light bands at different locations by using gel cutting
20 tips (Cleaver Scientific, UK). The selected bands were excised from the polyacrylamide gel, re-
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21 suspended in 20 µL sterilized UltraPureTM DNase/RNase-Free Distilled Water (Invitrogen, USA),

22 and stored overnight at 4 oC. The eluted DNA products were used as the template for re-
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23 amplification for sequencing analysis. The DNA sequences were compared against the GenBankTM
24 database and the Basic Local Alignment Search Tool (BLAST) for the closest matches.
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26 2.4.3. Microbial diversity
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28 The richness index (R) was used to characterize the microbial diversity and can be expressed as R =
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29 (S – 1) / ln N, where S is the total number of bacterial species in a sample and N is the total species
30 (Gao et al., 2014).
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32 3. Results and discussion
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34 3.1. Effect of aged sludge bioaugmentation on the toxic organic compounds biodegradation
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36 Figure 1 compares the batch-scale biodegradation rates of the target toxic organic compounds of the
37 seed sludge, 10%, 20%, and 30% (v/v) aged sludge bioaugmentation. The result showed that the
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1 biodegradation rate was positively correlated to the aged sludge ratio. The biodegradation rates were
2 0.012 – 0.032 h-1, 0.018 – 0.066 h-1, and 0.022 – 0.078 h-1 under the 10%, 20%, and 30% (v/v) aged
3 sludge bioaugmentation, respectively, compared to 0.010 – 0.028 h-1 without aged sludge.
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5 In this research, the 20% bioaugmentation ratio (v/v) was adopted because it could efficiently
6 degrade the target toxic organic compounds, relative to at the 10% ratio. Meanwhile, at 30%, the
7 biodegradation performance increased at a decreasing rate.

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9 Insert Figure 1

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11 3.2. Two-stage AS systems and treatment efficiency

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13 The results showed that aged sludge bioaugmentation enhanced the efficiency of organics and
14 nitrogen removal, especially under condition 1 where the BOD, COD, TOC, TKN, NH3-N, and TN
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removal efficiencies were 96%, 93%, 95%, 86%, 78%, and 84%, respectively. Under condition 2,
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16 the bioaugmented system outperformed the non-bioaugmented one, with the BOD, COD, TOC,
17 TKN, NH3-N, and TN treatment efficiencies of 96%, 95%, 96%, 80%, 90%, and 79%, respectively.
18 However, the treatment efficiency of the bioaugmented system under condition 3, even though
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19 higher than the non-bioaugmented one, was lower than under conditions 1 and 2. Under condition 3,
20 the BOD, COD, TOC, TKN, NH3-N, and TN treatment efficiencies were 92%, 87%, 91%, 76%,
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21 70%, and 72%, respectively. The efficient removal of organics and nitrogen was attributable to the
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22 abundant and diverse bacterial species in the aged sludge, especially nitrifying bacteria (Figure 3).
23 However, the effectiveness of aged sludge bioaugmentation in the removal of organics and nitrogen
24 was reduced when the compounds concentrations in the influent increased.
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26 In the non-bioaugmented system, under condition 1, the BOD, COD, TOC, TKN, NH3-N, and TN
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27 removal efficiencies were 96%, 93%, 95%, 81%, 78%, and 76%, respectively. Under condition 2,
28 the BOD, COD, TOC, TKN, NH3-N and TN treatment efficiencies were 96%, 93%, 95%, 78%,
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29 64%, and 74%, respectively. Under condition 3, the corresponding removal efficiencies were 90%,
30 92%, 89%, 75%, 77%, and 74%, respectively. Supplementary Figures 2 and 3 demonstrate the
31 treatment performance of the bioaugmented and non-bioaugmented systems under conditions 1, 2,
32 and 3, respectively.
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34 As shown in Table 2, the aerobic environment was conducive to the toxic organic compounds
35 biodegradation as the compounds were substantially degraded. As previously stated, both systems
36 were operated in parallel, with three levels of concentrations of organics, nitrogen and toxic organic
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1 compounds in the influent (conditions 1, 2, and 3). The purpose of the elevated concentrations was
2 to assess and compare the biodegradation performance of the bioaugmented system under various
3 conditions. Under condition 1, the BOD, COD, and TOC treatment efficiencies of the bioaugmented
4 and non-bioaugmented systems were similar. However, the BPA, 2,6-DTBP, DBP, DEHP, CBZ,
5 DCF, and DEET removal efficiencies of the bioaugmented system (90%, 89%, 86%, 77%, 85%,
6 75%, and 81%) were higher than those of the non-bioaugmented system (75%, 68%, 60%, 63%,
7 71%, 75%, and 65%).

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9 Under condition 2, the toxic organic compounds biodegradation efficiency of the bioaugmented

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10 system was lower than under condition 1. Under condition 2, the bioaugmented system could
11 remove 70-82% of the non-complex structure compounds (BPA, 2,6-DTBP, DBP, CBZ) and 70-

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12 74% of the complex structure compounds (DEHP, DCF, DEET). On the other hand, the treatment
13 efficiencies of the non-bioaugmented system were in the range of 47-64%. Under condition 3, the
14 toxic organic compounds biodegradation efficiencies of the bioaugmented and non-bioaugmented

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15 systems were reduced to 50-67% and 32-51%, respectively (Table 2).
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17 The bacterial abundance and diversity in the aged sludge improved the removal efficiency of toxic
18 organic compounds. However, the effectiveness of aged sludge bioaugmentation decreased as the
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19 compounds concentrations in the influent increased.

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21 Insert Table 2
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23 3.3. Bacterial abundance and bacterial community
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25 In Figure 2a, the DNA numbers of total bacteria, HNB, AOB, and NOB in the aged sludge were 2.43
26 x 1010, 4.53 x 106, 5.50 x 105, and 3.29 x 104 copies mL-1, respectively, and those in the seed sludge
(day 0) were 3.56 x 1010, 2.79 x 103, 1.72 x 102, and 1.32 x 102 copies mL-1, respectively. The
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28 important bacterial groups, including HNB, AOB, and NOB, were abundant in the aged sludge.
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30 Figure 2b shows total bacteria, AOB, NOB, and HNB in the anoxic and aerobic tanks of the
31 bioaugmented system under the three conditions (copies mL-1). Under condition 1, total bacteria
32 were 1.83 x 1012 and 2.06 x 1012 in the anoxic (an) and aerobic (ae) tanks; AOB 6.14 x 104 (an) and
33 6.41 x 104 (ae); NOB 8.82 x 103 (an) and 6.29 x 103 (ae); and HNB 2.12 x 108 (an) and 2.15 x 108
34 (ae).
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1 Under condition 2, total bacteria were 2.43 x 1012 (an) and 2.22 x 1012 (ae); AOB 8.14 x 104 (an) and
2 8.15 x 104 (ae); NOB 8.03 x 103 (an); and 9.07 x 103 (ae); HNB 1.33 x 108 (an) and 1.15 x 109 (ae).
3 Under condition 3, total bacteria were 1.91 x 1012 (an) and 2.13 x 1012 (ae); AOB 4.70 x 105 (an) and
4 5.42 x 105 (ae); NOB 1.39 x 104 (an) and 1.59 x 104 (ae); HNB 2.93 x 108 (an); and 1.16 x 108 (ae).
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6 As seen in Figure 2b, the bacterial abundance in the anoxic and aerobic tanks were mostly similar
7 under the same concentration conditions. This was attributable to the sludge recirculation from the

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8 sedimentation tank to the anoxic tank to maintain the anoxic condition and MLSS in the system as
9 well as the denitrification-nitrification reaction.

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11 Figure 2c shows total bacteria, AOB, NOB, and HNB in the anoxic and aerobic tanks of the non-

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12 bioaugmented system under the three conditions (copies mL-1): Under condition 1, total bacteria
13 were 5.84 x 1011 (an) and 5.40 x 1011 (ae); AOB 2.69 x 102 (an) and 2.42 x 102 (ae); NOB 3.01 x 102
14 (an) and 3.14 x 102 (ae); and HNB 1.99 x 103 (an) and 8.17 x 102 (ae).

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16 Under condition 2, total bacteria were 6.19 x 1011 (an) and 5.49 x 1011 (ae); AOB 3.54 x 102 (an) and
17 3.06 x 102 (ae); NOB 3.06 x 102 (an) and 4.23 x 102 (ae); and HNB 1.68 x 104 (an) and 1.41 x 104
18 (ae). Under condition 3, total bacteria were 4.28 x 1011 (an) and 4.78 x 1011 (ae); AOB 2.78 x 102
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19 (an) and 2.78 x 102 (ae); NOB 2.95 x 102 (an) and 2.67 x 102 (ae); and HNB 1.19 x 104 (an) and 5.47
20 x 103 (ae).
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22 As seen in Figure 2c, the bacterial abundance in the non-bioaugmented system was noticeably lower
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23 than in the bioaugmented one. This was probably attributable to limited nitrifying bacteria in the
24 non-bioaugmented system, resulting in less abundance of other bacterial species in the system.
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25
26 Insert Figure 2
27
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28 Figures 3a-c illustrate the DGGE profiles of the bioaugmented, non-bioaugmented systems and the
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29 seed and aged sludge. The source of the seed sludge was a local treatment plant treating low
30 compounds concentrations wastewater (BOD<500 mg L-1, COD<1000 mg L-1, and NH3-N<40 mg L-
1
31 ), so the seed sludge contained fewer bacterial communities than the aged sludge. However, over
32 the course of the operation, the bacterial communities in the sludge of the non-bioaugmented system
33 became more diverse (Figure 3b). The richness index of the non-bioaugmented system was in the
34 range of 1.74-3.78 (Table 3). The bacterial communities in the bioaugmented system were more
35 increasingly diverse (Figure 3a), including HNB (Bacillus, Pseudomonas, and Sphingomonas) and
36 NB (Nitrosococcus, Nitrosomonas, Nitrobacter, and Nitrospira), which are crucial to the toxic
 12
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1 organic compounds biodegradation (Xu et al., 2016; Kruglova et al, 2017). The richness index of the
2 bioaugmented system was in the range of 3.49-6.69 (Table 3).
3
4 According to Merkey et al. (2009) and Ni et al. (2011), AOB, which is abundant in aged sludge,
5 favored the inorganic compounds in wastewater and released soluble microbial products (SMP),
6 which are the carbon organic compounds beneficial for the growth of HB. For this reason, the total
7 organic compound (TOC) concentrations in the anoxic and aerobic tanks of the bioaugmented

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8 system were higher than in the non-bioaugmented one. In addition, NB in the bioaugmented system
9 promoted the growth of HB and HNB, which are the important bacterial groups in the toxic organic

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10 compounds biodegradation. HB and HNB produced many important degrading enzymes,
11 particularly phenol monooxygenase, which is an enzyme that converts phenols into an intermediate

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12 compound (i.e., catechol) and acts as the phenol-degradation rate-limiter (Chen, 2003; Zhang et al.,
13 2004). The HB and HNB species that produce phenol monooxygenase are Pseudomonas sp.,
14 Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas syringae.

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15
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16 For PAE biodegradation, the phthalate-degrading enzymes esterase and phthalate dioxygenase were
17 produced by the bacterial species Bacillus subtilis (Peerzada et al., 2006), Pseudomonas fluorescens,
18 Pseudomonas putida (Shaw et al., 2006; Rehdorf et al., 2012), Burkholderia sp., Bacillus
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19 multivorans, Ralstonia pickettii, and Pseudomonas putida (Boonnorat et al., 2014a) in the HB and
20 HNB groups. These phthalate-degrading bacterial species were abundant in the bioaugmented
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21 system. Table 3 tabulates the bacterial species in the seed sludge, aged sludge, the bioaugmented and
22 non-bioaugmented AS systems as well as the richness index under conditions 1, 2, and 3.
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23
24 Insert Figure 3
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25 Insert Table 3
26
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27 According to Fernandez-Fontaina et al. (2016) and Park et al. (2017), the enzyme AMO enhanced
28 the biodegradation of toxic organic or aromatic compounds of an AOB-abundant treatment system.
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29 Similarly, the abundant AOB in the bioaugmented system enhanced the toxic organic compounds
30 biodegradation efficiency. In addition, the biodegradation performance in the aerobic environment
31 was higher, compared with in the anoxic environment, due to ample supplies of oxygen. Essentially,
32 the aged sludge bioaugmentation could enhance the toxic organic compounds biodegradation and
33 wastewater treatment efficiency of the two-stage AS system. Despite the limited treatment efficiency
34 under condition 3, aged sludge is a cost-effective bioaugmenter that could improve the treatment
35 efficiency of the biological AS system.
36
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1 4. Conclusion
2
3 This research investigated the toxic organic compounds biodegradation efficiency of the two-stage
4 AS systems with (bioaugmented) and without (non-bioaugmented system) aged sludge
5 bioaugmentation. The bioaugmented and non-bioaugmented systems were operated in parallel, with
6 three levels (low, moderate, and high) of concentrations of organics, nitrogen, and toxic organic
7 compounds in the influent (conditions 1, 2, and 3). The results showed that both systems could

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8 efficiently remove BOD, COD, and TOC. Nevertheless, the toxic organic compounds
9 biodegradation efficiency of the bioaugmented system was higher than that of the non-bioaugmented

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10 one. The aged sludge bioaugmentation enhanced the biodegradation efficiency under conditions 1
11 and 2. However, the treatment efficiency of the bioaugmented system was reduced under condition

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12 3. The qPCR and DGGE results indicated that HB, HNB, and NB (both AOB and NOB) were
13 abundant in the aged sludge. As a result, the bioaugmented system could achieve the higher
14 biodegradation and wastewater treatment efficiency than the non-bioaugmented one. Despite the

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15 limited biodegradation efficiency of the bioaugmented system under condition 3, aged sludge is an
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16 operationally-viable and environmentally-friendly bioaugmenter that could improve the treatment
17 efficiency of the biological AS system.
18
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19 Acknowledgements
20
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21 The authors would like to extend deep gratitude to the Institute of Research and Development (IRD)
of Rajamangala University of Technology Thanyaburi (RMUTT) for the financial sponsorship
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22
23 through the Funding for Young Researcher Program (Grant Numbers NRF04066008 and 8/2560)
24 and The Research Grant for New Scholar (MRG61) by The Thailand Research Fund (TRF) – (Thai
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25 Government Fund). Sincere appreciation also goes the Biological Engineering Program, Faculty of
26 Engineering, King Mongkut’s University of Technology Thonburi (KMUTT) for the technical
27 assistance.
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28
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29
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Table 1 The characteristics of the mixed influent with three levels (low, moderate, high) of concentrations of organics, nitrogen and toxic organic compounds
(conditions 1, 2, and 3)
Concentration a

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Parameters Unit
Condition 1 (days 0-77) b Condition 2 (days 78-161) b Condition 3 (days 162-245) c
Average (SD) Average (SD) Average (SD)
Water qualities

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pH - 7 – 7.2 6.9 – 7.2 6.8 – 7.1
BOD mg L-1 860 (90) 1540 (90) 3250 (180)
COD mg L-1 1300 (120) 2800 (120) 5800 (120)

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TOC mg L-1 420 (90) 830 (50) 1630 (50)
TKN mg L-1 210 (60) 330 (90) 580 (110)
NH3-N mg L-1 100 (30) 170 (40) 240 (50)
TN mg L-1 240 (20) 370 (30) 620 (40)

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Toxic organic compounds
BPA µg L-1 53 (10) 78 (12) 109 (20)

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2,6-DTBP µg L-1 19 (5) 28 (5) 48 (11)
DBP µg L-1 23 (5) 32 (8) 49 (10)
DEHP µg L-1 22 (6) 31 (7) 56 (13)
µg L-1

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CBZ 7 (3) 17 (4) 24 (5)
DCF µg L-1 4 (2) 10 (3) 12 (3)
DEET µg L-1 81 (26) 105 (22) 166 (42)
a
The number of samples under each condition was 12.

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b
Mixed influent (leachate and agriculture wastewater) in ratio of 1:1 (v/v) with RO water dilution
c
Mixed influent (leachate and agriculture wastewater) in ratio of 1:1 (v/v)

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Table 2 Toxic organic compounds concentrations in the anoxic, aerobic and effluent tanks and the biodegradation efficiency (20% aged sludge, v/v) under conditions 1, 2, and 3

Compounds Unit Concentration *

Condition 1 (days 0-77) Condition 2 (days 78-161) Condition 3 (days 162-245)

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Anoxic Aerobic Effluent Anoxic Aerobic Effluent Anoxic Aerobic Effluent

Bioaugmented System
BPA µg L-1 42 (9) 11 (2) 5 (3) † 66 (10) 35 (6) 14 (3) 95 (19) 70 (18) 37 (12)

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90 % ‡ 82 % 66 %
2,6-DTBP µg L-1 14 (3) 4 (3) 2 (2) 23 (5) 9 (3) 5 (4) 43 (13) 22 (8) 19 (8)
89 % 80 % 60 %

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DBP µg L-1 19 (4) 8 (3) 3 (2) 27 (7) 12 (2) 9 (4) 43 (10) 24 (4) 16 (5)
86 % 70 % 67 %
DEHP µg L-1 17 (6) 6 (3) 5 (3) 27 (6) 11 (2) 8 (1) 52 (13) 36 (12) 28 (8)
77 % 74 % 50 %

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CBZ µg L-1 4 (2) 1 (1) 1 (1) 13 (3) 8 (3) 5 (3) 22 (5) 16 (6) 10 (5)
85 % 70 % 58 %

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DCF µg L-1 2 (1) 1 (1) 1 (1) 8 (3) 5 (3) 3 (2) 11 (3) 7 (2) 6 (3)
75 % 70 % 50 %
DEET µg L-1 67 (27) 24 (12) 15 (7) 90 (15) 29 (16) 28 (8) 149 (46) 87 (33) 70 (29)

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81 % 73 % 57 %

Non-bioaugmented System
BPA µg L-1 43 (10) 23 (5) 13 (4) † 68 (10) 35 (6) 29 (5) 98 (19) 70 (18) 65 (16)

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75 % ‡ 62 % 40 %
2,6-DTBP µg L-1 15 (3) 8 (2) 6 (2) 24 (5) 13 (3) 10 (4) 45 (12) 27 (8) 24 (8)

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68 % 64 % 50 %
DBP µg L-1 19 (5) 12 (4) 9 (2) 28 (7) 18 (5) 15 (5) 45 (10) 26 (4) 24 (5)
60 % 53 % 51 %
DEHP µg L-1 18 (7) 10 (4) 8 (4) 28 (6) 15 (3) 13 (3) 52 (14) 40 (12) 38 (11)
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63 % 58 % 32 %
CBZ µg L-1 4 (2) 2 (1) 2 (1) 14 (3) 11 (3) 10 (3) 22 (5) 17 (7) 16 (6)
71 % 41 % 33 %
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DCF µg L-1 3 (1) 2 (1) 1 (1) 8 (3) 6 (3) 5 (3) 11 (3) 9 (3) 8 (3)
75 % 50 % 33 %
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DEET µg L-1 69 (26) 35 (14) 28 (14) 93 (17) 59 (16) 55 (16) 153 (45) 118 (41) 108 (41)
65 % 47 % 34 %
*
The number of samples under each condition was 12.
†
Average value (SD)
‡
Total biodegradation (%)
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Table 3 Bacterial communities in the seed sludge, aged sludge, the bioaugmented (20% aged sludge, v/v), non-bioaugmented AS systems and richness index under conditions 1, 2,
and 3

Bacterial species Band No. Seed Aged Bioaugmented System Non-bioaugmented System
(similarity more than 90%) Cond. 1 Cond. 2 Cond. 3 Cond. 1 Cond. 2 Cond. 3

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An Ae An Ae An Ae An Ae An Ae An Ae
Heterotrophic bacteria (HB)
Agrobacterium sp. 9a, 3c + + + +
Burkholderia sp. 3a, 8a, 8b + + + + + + + + + + +

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Candidatus Endolissoclinum patella 17a, 13b + + + +
Dechloromas aromatic 2a, 2b + + + +
Enterobacter sp. 27a, 19b + + + + + +

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Flavobacterium sp. 1a, 1b + + + +
Pectobacterium wasabiae 31a, 32a, 20b, 21b + + + + +
Ralstonia pickettii 34a, 14c + +
Rhodopseudomonas palustris 33a, 13c + + +

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Sphingomonas sp. 19a, 15b, 9c + + + + + + +
Streptococcus sinensis 20a + +

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Streptococcus sp. 23a + + + +
Xanthomonas albillineans 21a + + +
Xanthomonas sp. 22a + + + +

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Heterotrophic nitrifying bacteria (HNB)
Bacillus cereus 11a, 9b, 4c + + + + + + + + + + +
Bacillus sp. 4a, 3b, 4b, 1c + + + + + + + + + +
Bacillus subtilis 5a, 5b + + + + + + + +

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Pseudomonas aeruginosa 12a, 5c + + +
Pseudomonas fluorescens 7a, 7b, 2c + + + + + + + + + +

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Pseudomonas putida 24a, 16b + + + + + + + +
Pseudomonas sp. 16a, 7c + + + + + + +
Pseudomonas syringae 6a, 6b + + + + + +
Thiobacillus denitrificans 10a + +
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Nitrifying bacteria (NB)
Ammonia oxidizing bacteria (AOB)
Nitrosococcus mobilis 13a, 10b, 6c + + + + + + + + + +
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Nitrosococcus sp. 28a, 12c + + + + + + +

Nitrosomonas eutropea 18a, 14b, 8c + + + + + + + + + + + + +
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Nitrosomonas sp. 26a, 18b, 10c + + + + + + + + + + + + +

Nitrite oxidizing bacteria (NOB)
Nitrobacter hamburgensis 14a + + + +
Nitrobacter alkalicus 15a, 12b + +
Nitrobacter sp. 25a, 17b, 11c + + + + + + + + + + + + + +
Nitrospira sp. 29 a, 30a + + + + + + + +

Richness index (R) 3.49 4.95 6.69 5.53 6.69 5.53 2.62 1.74 3.20 1.45 3.78 3.78
+ indicates the presence of DGGE band
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BPA 2,6-DTBP DBP

0 6 12 18 24 0 6 12 18 24 0 6 12 18 24
0.00 0.00 0.00
y = -0.0245x y = -0.0248x
R² = 0.7017 R² = 0.8589 y = -0.0284x
R² = 0.6366
-0.40 -0.40 -0.40

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y = -0.0259x
y = -0.0278x
-0.80 R² = 0.7491 -0.80 -0.80
ln (C/C0)

ln (C/C0)

ln (C/C0)
R² = 0.8434 y = -0.0326x
R² = 0.6966
y = -0.0665x
-1.20 R² = 0.9658 -1.20 y = -0.0527x -1.20

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y = -0.0631x
R² = 0.9715 R² = 0.9333
-1.60 -1.60 -1.60
y = -0.0787x y = -0.0683x y = -0.0737x
R² = 0.968 R² = 0.975 R² = 0.9186

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-2.00 -2.00 -2.00
Time (h) Time (h) Time (h)

DEHP CBZ DCF

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0 6 12 18 24 0 6 12 18 24 0 6 12 18 24
0.00 0.00 0.00
AN
y = -0.0216x y = -0.0105x
-0.20 y = -0.0203x
R² = 0.4519 R² = 0.7514
-0.20 R² = 0.829
-0.40
-0.20
-0.40
ln (C/C0)
ln (C/C0)

ln (C/C0)

-0.60
y = -0.0126x y = -0.0217x
M

y = -0.0238x
R² = 0.8273 R² = 0.732
-0.80 R² = 0.486
-0.60
y = -0.0407x -0.40 y = -0.0184x y = -0.033x
-1.00 R² = 0.9444
R² = 0.8931 R² = 0.9163
-0.80
y = -0.0376x
D

-1.20 y = -0.046x y = -0.0223x

R² = 0.8712 R² = 0.971 R² = 0.9612
-1.40 -0.60 -1.00
Time (h) Time (h) Time (h)
TE

DEET
0 6 12 18 24
EP

0.00 Seed sludge (control)

y = -0.0159x
R² = 0.7942 Seed sludge + 10 % aged sludge
-0.20
Seed sludge + 20 % aged sludge
y = -0.0258x
C

-0.40 R² = 0.7794 Seed sludge + 30 % aged sludge

ln (C/C0)

Linear (Seed sludge (control))

AC

-0.60 y = -0.0352x
R² = 0.9968 Linear (Seed sludge + 10 % aged sludge)

-0.80 y = -0.041x Linear (Seed sludge + 20 % aged sludge)

R² = 0.9422
Linear (Seed sludge + 30 % aged sludge)
-1.00
Time (h)

Figure 1 Biodegradation rates of toxic organic compounds of the seed sludge, 10%, 20%, and
30% (v/v) aged sludge bioaugmentation in the aerobic environment
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1.00E+14
1.00E+12 Seed sludge Aged sludge
1.00E+10
Copies mL-1

1.00E+08
1.00E+06
1.00E+04

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1.00E+02
1.00E+00
Total bacteria amoA gene AOB Nitrobacter Nitrospira Total Heterotrophic
heterotrophic nitrifying

RI
bacteria bacteria

(a)

SC
1.00E+14
1.00E+12 An. - condition 1 Ae. - condition 1
1.00E+10 An. - condition 2 Ae. - condition 2
Copies mL-1

U
1.00E+08
An. - condition 3 Ae. - condition 3
1.00E+06
AN
1.00E+04
1.00E+02
1.00E+00
Total bacteria amoA gene AOB Nitrobacter Nitrospira Total Heterotrophic
M

heterotrophic nitrifying
bacteria bacteria

(b)
D
TE

1.00E+14
1.00E+12
An. - condition 1 Ae. - condition 1
Copies mL-1

1.00E+10 An. - condition 2 Ae. - condition 2

1.00E+08
An. - condition 3 Ae. - condition 3
EP

1.00E+06
1.00E+04
1.00E+02
1.00E+00
Total bacteria amoA gene AOB Nitrobacter Nitrospira Total Heterotrophic
C

heterotrophic nitrifying
bacteria bacteria
AC

(c)

Figure 2 Bacterial abundance: (a) the seed and aged sludge, (b) the bioaugmented system (20%,
v/v), (c) the non-bioaugmented system
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PT
RI
U SC
AN
M
D
TE
C EP
AC

Figure 3 DGGE profiles of: (a) the bioaugmented system (20%, v/v), (b) the non-bioaugmented system, (c) the seed and aged sludge,
where An1, An2, An3, Ae1, Ae2, Ae3 are the anoxic (an) and aerobic (ae) mixed liquors under conditions 1, 2, and 3
 ACCEPTED MANUSCRIPT
Highlights
• Aged sludge bioaugmentation enhances the toxic organic compounds biodegradation
of the two-stage activated sludge system.
• Aged sludge promotes the growth of heterotrophic bacteria (HB) and heterotrophic
nitrifying bacteria (HNB).
• The diversity and abundance of HB, HNB, and nitrifying bacteria (NB) enhance the

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toxic organic compounds biodegradation.

RI
U SC
AN
M
D
TE
C EP
AC