Science of the Total Environment 724 (2020) 138275

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Science of the Total Environment

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Effect of leachate effluent from activated sludge and membrane

bioreactor systems with acclimatized sludge on plant seed germination
Alongkorn Kanyatrakul a,1, Apichai Prakhongsak a,1, Ryo Honda b, Supaporn Phanwilai c,
Chairat Treesubsuntorn d, Jarungwit Boonnorat a,⁎
a
Department of Environmental Engineering, Faculty of Engineering, Rajamangala University of Technology Thanyaburi (RMUTT), Klong 6, Pathum Thani 12110, Thailand
b
Faculty of Geosciences and Civil Engineering, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
c
Department of Environmental Engineering, Faculty of Engineering, Kasetsart University, Jatujak, Bangkok 10900, Thailand
d
Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi (KMUTT), Bangkhuntien, Bangkok 10150, Thailand

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• AS and MBR with acclimatized sludge

efficiently remove micropollutants.
• MBR with acclimatized sludge effi-
ciently degrades micropollutants in
landfill-leachate influent of high con-
centration.
• MBR with acclimatized sludge achieves
very high germination rate and germi-
nation seed index.
• MBR technology can be potentially ap-
plied to water reclamation.

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

Article history: This research comparatively investigates the effect of landfill leachate effluent of two biological treatment
Received 8 December 2019 schemes on germination of Lactuca sativa and Vigna radiata. The treatment schemes are two-stage activated
Received in revised form 29 February 2020 sludge (AS) and two-stage membrane bioreactor (MBR) systems with acclimatized seed sludge. The AS and
Accepted 26 March 2020
MBR are operated under two concentrations of landfill leachate influent: moderate (condition 1) and elevated
Available online 27 March 2020
(condition 2). The results show that, under condition 1, the AS and MBR efficiently remove 80–96% of organic
Editor: Charlotte Poschenrieder compounds and nutrients and 81–100% of harmful micropollutants. Under condition 2 with elevated influent
concentration, MBR is more effective in biodegrading micropollutants than the AS system. The germination
Keywords: rate (GR) and germination seed index (GSI) of L. sativa and V. radiata germinated with AS and MBR effluent
Seed germination from condition 1 are 100% and 1.29–1.56. Under condition 2, the GR and GSI with AS effluent are reduced to
Landfill leachate 80% and 0.65–0.77, while those with MBR effluent are 100% and 1.27–1.38. Quantitative real-time polymerase
Activated sludge (AS) chain reaction (qPCR) analysis indicates that the bacterial community in the MBR is more abundant than in
Membrane bioreactor (MBR) the AS, especially ammonia oxidizing bacteria, Nitrobacter, and Nitrospira, which aid heterotrophic bacteria in
biodegradation of micropollutants and promote the growth of heterotrophs. The bacterial abundance and com-
munity composition render the MBR scheme more operationally suitable for elevated landfill-leachate influent
concentrations. By comparison, the MBR system is more effective in removal of micropollutants than the AS, as
evidenced by higher GR and GSI. The technology also could potentially be applied to water reclamation. A lack
of technological and financial resources in many developing countries nevertheless precludes the adoption of

⁎ Corresponding author.
E-mail address: jarungwit_b@rmutt.ac.th (J. Boonnorat).
1
These authors contributed equally to this work as first co-authors.

https://doi.org/10.1016/j.scitotenv.2020.138275
0048-9697/© 2020 Elsevier B.V. All rights reserved.
2 A. Kanyatrakul et al. / Science of the Total Environment 724 (2020) 138275
MBR despite higher pollutant removal efficiency. An alternative solution is the use of acclimatized seed sludge in
AS system to enhance treatment efficiency, especially in influent with low concentrations of micropollutants. In
addition, the seed germination results suggest the possibility of water reuse in agriculture.
1. Introduction
Landfill leachate contains a wide range of micropollutants of varying
concentrations and toxicities. Several micropollutants in landfill leach-
ate are difficult to remove using conventional treatment systems due
to their complex molecular structures, such as phenols, phthalates,
and hydrocarbon compounds. Even landfill leachate with low concen-
trations of micropollutants could adversely impact the ecosystem (Asif
et al., 2018; Shao et al., 2019). Some micropollutants, even in trace
amounts, are harmful to human health (Schwarzenbach et al., 2006)
and can pollute natural waterways which are habitat of aquatic animals
(Boonnorat et al., 2017). According to Huang et al. (2010),
micropollutants present in landfill leachate, including bisphenol A
(BPA) and di-(ethylhexyl)-phthalate (DEHP), are carcinogenic and haz-
ardous to human health.
Several wastewater treatment technologies have been proposed for
removal of micropollutants and harmful substances before discharge
into natural waterways or reuse. Examples of such technologies are ad-
vanced oxidation process (AOP) with TiO2 photocatalysis (Hassan et al.,
2016), ozone-based process (Gautam et al., 2019), chemical-based ion-
exchange system to remove toxic metals during biological treatment
(Robinson, 2017), activated sludge (AS) augmented with acclimatized
bacterial sludge to enhance micropollutant biodegradation in landfill
leachate (Boonnorat et al., 2018), hybrid moving bed biofilm reactor-
membrane bioreactor (MBBR-MBR) (Jiang et al., 2018), and use of
laccase enzyme in MBR to enhance micropollutant biodegradation
(Asif et al., 2018).
The aforementioned micropollutant biodegradation technologies
fall into two categories: conventional treatment, e.g., activated sludge;
and advanced schemes, e.g., MBR. Indeed, existing research on
micropollutant removal predominantly focuses on MBR technology
(Xue et al., 2010; Liu et al., 2017; Song et al., 2018). The advantages of
MBR include higher treatment efficiency, especially polluted wastewa-
ter; and system compactness. Meanwhile, the shortcomings of the tech-
nology are high investment and maintenance costs and requirement for
skilled manpower.
In reality, several areas, especially in developing countries, lack
technological and financial resources, preventing them from adopting
the MBR technology. A practical alternative is the use of acclimatized
sludge bioaugmentation in AS system to enhance treatment efficiency,
especially in influent with low concentrations of micropollutants
(Boonnorat et al., 2018). As a result, this research utilizes acclimatized
sludge (N1 year old) as seed sludge in AS and MBR systems operated
under the same conditions. The treatment performance, particularly
micropollutant biodegradation, of both systems treating two concentra-
tions of leachate influent is subsequently compared. Furthermore, seed
germination experiments are carried out using effluent from both
treatment systems to explore the possibility of water reuse in
agriculture.
The bacterial community in biological wastewater treatment
systems plays an essential role in the removal efficiency of
micropollutants. Boonyaroj et al. (2017) achieved 65% and 70% removal
efficiency for BPA and 2,6-di-tert-butyl-phenol (BHT) by enriching nitri-
fying bacteria in the MBR system. Boonnorat et al. (2019) employed
acclimatized sludge augmentation to enhance micropollutant biodegra-
dation of AS system and reported that acclimatized sludge improves the
AS effluent quality, especially in low-micropollutant concentration
environment.
© 2020 Elsevier B.V. All rights reserved.
In addition, the advanced treatment schemes (e.g., AOPs, MBR, and
enzymatic-MBR) and biological treatment systems augmented with ac-
climatized sludge containing effective bacterial groups efficiently re-
move harmful micropollutants in the landfill leachate. Currently, there
exists no study on effects of effluent from advanced and biological treat-
ment systems on seed germination. Specifically, existing research on
seed germination focuses on contaminated water or diluted wastewater
(Pan et al., 2013; Li et al., 2017). In other words, there is no research
work on plant seed germination using treated wastewater or effluent.
This current research thus examines the effect of landfill leachate efflu-
ent on seed germination.
Lactuca sativa (lettuce) is a model plant species for phytotoxicity
tests and has been recommended by international organizations for
determination of the phytotoxic effects of toxic substances. Phyto-
toxicity tests using L. sativa are straightforward, fast, reliable, and
inexpensive as no elaborate equipment is required (Lyu et al.,
2018). Additionally, Kim et al. (2018) also examined the effect of
micropollutants, particularly BPA, on Viana radiata (mung bean).
Both plant species (L. sativa and V. radiata) are commonly used in
phytotoxicity evaluation of harmful chemical compounds in the en-
vironment, including landfill leachate micropollutants (Boonnorat
et al., 2019; Kim et al., 2018).
Due to lack of research on the effects of treated wastewater on seed
germination, this research investigates the effect of landfill leachate ef-
fluent of two biological treatment schemes on germination of Lactuca
sativa and Vigna radiata seeds. The treatment schemes are two-stage
AS and two-stage MBR with acclimatized seed sludge. The AS and
MBR are operated under two concentrations of landfill leachate influ-
ent: moderate (condition 1; days 0–42) and elevated (condition 2;
days 43–84). L. sativa and V. radiata seeds are used due to their fast re-
action to chemical exposure (Rede et al., 2016). The target
micropollutants are bisphenol A (BPA), 2,6-di-tert-butyl-phenol
(2,6-DTBP), di-ethyl-phthalate (DEP), di-butyl-phthalate (DBP), di-
(ethylhexyl)-phthalate (DEHP), and N,N-diethyl-m-toluamide
(DEET). The plant seeds are germinated with RO water (the control),
AS effluent (of conditions 1 and 2), and MBR effluent of both condi-
tions. The results are measured in terms of germination rate and ger-
mination seed index.
2. Materials and methods
2.1. Two-stage activated sludge (AS) and two-stage membrane bioreactor
(MBR) systems
In this research, the experiments were parallelly carried out by using
two-stage AS and two-stage MBR. The membrane was of hollow fiber
module with a 0.4 μm pore size. Two acrylic tanks with a working vol-
ume of 10 L each were used as anoxic and aerobic tanks. The seed sludge
in the experimental AS and MBR was acclimatized sludge treating land-
fill leachate for more than a year (Boonnorat et al., 2018). Both treat-
ment schemes were operated under the mixed liquor suspended
solids (MLSS) of acclimatized seed sludge of 5 g/L and the hydraulic re-
tention time (HRT) of 24 h. Over the course of the operation, the sludge
in the aerobic tank was recirculated into the anoxic tank to maintain the
system's nitrification-denitrification (Fig. 1).
This research used a mixture of young and old landfill leachate as the
influent. The young leachate was obtained from municipal waste col-
lected daily by garbage trucks, and the old leachate was from oxidation
 A. Kanyatrakul et al. / Science of the Total Environment 724 (2020) 138275 3

Fig. 1. The experimental framework of this study.

ponds at the landfill site. The municipal landfill site has been in opera- Table 1 tabulates the characteristics of both influent concentrations:
tion over two decades, and food waste, plastics, and household waste moderate (condition 1; days 0–42) and elevated (condition 2; days
account for the largest proportion of the waste at the landfill site. 43–84).

Table 1
Landfill leachate characteristics of two-stage AS and MBR systems.

Parameters Unit Influent Treatment system

Two-stage AS Two-stage MBR

Anoxic Aerobic Effluent Anoxic Aerobic Effluent

pH - 6.6 – 7.4a 7 – 7.4 7.1 – 7.4 7.2 – 7.5 6.9 – 7.1 7 – 7.2 7.1 – 7.3
6.3 – 7.2b 6.9 – 7.1 7 – 7.3 7.1 – 7.5 6.8 – 7.2 7 – 7.4 7 – 7.4
BOD mg/L 1100 (570) 520 (120) 250 (80) 22 (15), 95%c 450 (110) 87 (25) 12 (8), 98%
2900 (480) 1240 (340) 410 (50) 45 (30), 98%d 800 (160) 210 (70) 25 (10), 99%
COD mg/L 3200 (360) 1400 (130) 610 (95) 100 (40), 96% 1100 (80) 350 (68) 65 (40), 98%
5800 (610) 2800 (110) 930 (60) 380 (70), 93% 2400 (120) 420 (50) 220 (50), 96%
TOC mg/L 840 (64) 224 (42) 105 (27) 17 (5), 98% 238 (25) 61 (14) 8 (4), 99%
1240 (57) 618 (29) 295 (16) 26 (9), 98% 480 (12) 117 (21) 13 (5), 99%
TKN mg/L 114 (24) 75 (20) 36 (12) 25 (5), 78% 72 (14) 21 (6) 14 (8), 88%
220 (35) 164 (25) 72 (24) 37 (4), 83% 148 (19) 47 (11) 23 (6), 89%
NH3 mg/L 25 (12) 16 (4) 7 (3) 5 (2), 80% 17 (5) 6 (3) 4 (2), 84%
42 (15) 24 (3) 10 (4) 5 (3), 88% 20 (4) 8 (5) 5 (3), 88%
TN mg/L 127 (16) 86 (15) 42 (11) 21 (5), 83% 84 (13) 28 (17) 17 (4), 87%
250 (11) 187 (18) 81 (15) 42 (6), 83% 150 (16) 69 (22) 31 (7), 88%
PO3 mg/L 25 (8) 22 (4) 13 (8) 4 (2), 84% 20 (3) 9 (7) 4 (2), 84%
67 (9) 52 (9) 35 (7) 16 (5), 76% 42 (5) 29 (2) 8 (3), 88%
As ppm 0.018 (0.004) NA NA 0.013 (0.004) NA NA 0.011 (0.002)
0.024 (0.006) NA NA 0.015 (0.005) NA NA 0.012 (0.001)
Cd ppm 0.002 (0.001) NA NA ND NA NA ND
0.005 (0.003) NA NA 0.002 (0.001) NA NA 0.002 (0.001)
Ni ppm 0.004 (0.003) NA NA 0.003 (0.001) NA NA ND
0.010 (0.004) NA NA 0.006 (0.002) NA NA 0.004 (0.001)
Pb ppm 0.027 (0.010) NA NA 0.023 (0.013) NA NA 0.014 (0.005)
0.043 (0.022) NA NA 0.031 (0.015) NA NA 0.028 (0.003)

Number of samples of each condition is 7 and values are in average (SD).

NA denotes not analyzed.
ND denotes not detected (under instrumental detection limit).
a
denotes the influent concentration of condition 1 (days 0 – 42).
b
denotes the influent concentration of condition 2 (days 43 – 84).
c
denotes treatment efficiency (%) of condition 1.
d
denotes treatment efficiency (%) of condition 2.
4 A. Kanyatrakul et al. / Science of the Total Environment 724 (2020) 138275
2.2. Water quality and wastewater characteristics
Samples of influent, anoxic water, aerobic water, and effluent of AS
and MBR systems were collected and analyzed on a weekly basis ac-
cording to the Standard Methods for the Examination of Water and
Wastewater (APHA, 2005). The micropollutants were determined in
liquid phase by using solid phase extraction (SPE) technique and char-
acterized by gas chromatography–mass spectrometry (GC–MS).
In the analysis, the SPE tubes (VertipakTM-C18) were used for
micropollutant extraction. The tubes were first cleaned with 10 mL
methanol (MeOH) and then with 10 mL RO water before introducing
100 mL water samples. The tubes were left to settle and then eluted
with 10 mL MeOH. The elution fraction was evaporated with pure nitro-
gen gas and analyzed by GC–MS (Boonnorat et al., 2014). Heavy metals
were analyzed by inductively coupled plasma-optical emission spec-
trometry (ICP-OES) (A et al., 2017).
2.3. Bacterial community by quantitative real-time polymerase chain reac-
tion (qPCR)
Samples of anoxic and aerobic sludge of AS and MBR systems under
conditions 1 and 2 were extracted by DNA extraction kit (Qiagen,
Germany). qPCR was used to quantify total bacteria and nitrifying bac-
teria, including ammonia oxidizing bacteria (AOB), Nitrobacter, and
Nitrospira. The primers and the quantification protocol followed
Boonnorat et al. (2017).
2.4. Seed germination test
L. sativa and V. radiata seeds germinated with AS and MBR effluents
were performed in triplicate under the same conditions
(e.g., temperature, relative humidity) in Petri dishes lined with tissue
paper sprayed with 1 mL of RO water (i.e., the control), 1 mL of AS efflu-
ent (of conditions 1 and 2), and 1 mL of MBR effluent (of conditions 1 and
2). Each Petri dish contained 20 seeds of L. sativa or V. radiata. The obser-
vation was undertaken after 12, 24, 36, and 48 h. The seed-germination
temperature and relative humidity were 30 °C and 80%, and the seeds
were commercial plant seeds. The germination rate, which is the
Fig. 2. Micropollutant characteristics of condition 1 (A, B, C) and condition 2 (D, E, F), where A and D are influent, B and E are AS effluent, C and F are MBR effluent. *Indicates compounds
whose concentrations are presented in Table 2.
percentage of germinated seeds in relation to total number of seeds;
and the germination seed index are calculated by (Aguiar et al., 2016).
Germination rate ð%Þ ¼ TN=N  100
where TN is the total number of germinated seeds and N is the total
number of seeds in each dish.
Germination seed index = (N1 ∗ 1) + (N2 − N1) ∗ 1/
2 + (N3 − N2) ∗ 1/3 + …[Nx − (Nx−1)] ∗ 1/ywhere Nx is the number
of germinated seeds in a given period and y is total evaluation time
(i.e., y = 4, consisting of 12, 24, 36, and 48 h after seed planting).
3. Results and discussion
3.1. Treatment performance and micropollutant biodegradation of two-
stage AS and MBR
As seen in Table 1, under condition 1, the treatment performance of
two-stage AS and two-stage MBR with acclimatized seed sludge are
95–99% for organic compounds (BOD, COD, TOC) and 78–88% for nutri-
ent groups (N and P). Under condition 2 with elevated influent concen-
tration, the MBR with acclimatized sludge achieves higher treatment
efficiency for organic compounds (96–99%) and nutrient groups
(88–89%), in comparison with 93–98% and 76–88% of the AS with accli-
matized sludge. The influent (of conditions 1 and 2) contains low con-
centrations (0.004–0.043 ppm) of heavy metals (As, Cd, Ni, Pb).
However, some heavy metals are not detected in the effluent, including
Cd and Ni. By comparison, the heavy metal concentrations in the MBR
effluent are lower than in the AS effluent.
The characteristics of micropollutants in the influent and effluent of
conditions 1 and 2 are illustrated in (Fig. 2). The predominant
micropollutants are aromatic compounds and derivatives of plasticizers,
such as phenols and phthalates which are endocrine disrupting
chemicals that are harmful to human health and the environment
(Huang et al., 2010). In (Fig. 2), low micropollutant concentrations are
represented by blue color of varying shades and high concentrations
by yellow color of different shades. The result indicated that
 A. Kanyatrakul et al. / Science of the Total Environment 724 (2020) 138275 5

acclimatized sludge enhances the removal of compounds in the AS and
MBR systems, especially under condition 1 as evidenced by several dark
blue bands. Under condition 2 with elevated influent concentration, the
micropollutant biodegradation efficiency of MBR with acclimatized
sludge resembles that of condition 1, while the removal efficiency of
AS with acclimatized sludge slightly decreases.
Several micropollutants present in landfill leachate are endocrine
disrupting chemicals and carcinogenic compounds (Paxeus, 2000;
Kjeldsen et al., 2002; Slack et al., 2005; Michalowicz and Duda, 2007).
These micropollutants include bisphenol A (BPA), 2,6-di-tert-butyl-
phenol (2,6-DTBP), di-ethyl-phthalate (DEP), di-butyl-phthalate
(DBP), di-(ethylhexyl)-phthalate (DEHP), and N,N-diethyl-m-
toluamide (DEET) (Table 2). Under condition 1, AS and MBR efficiently
degrade the target compounds (82–100%), but under condition 2 MBR
is more effective in biodegrading micropollutants. Under condition 2,
the MBR biodegradation efficiencies for BPA, 2,6-DTBP, DEP, DBP,
DEHP, and DEET are 90%, 89%, 86%, 90%, 87%, and 82%, compared with
62%, 74%, 87%, 61%, 65%, and 45% of the AS system under the same influ-
ent condition. Specifically, the micropollutant biodegradation of AS is
significantly reduced (p b 0.05) as the influent concentration is in-
creased. This is not the case with MBR (p N 0.05), however.
In this research, the AS and MBR systems with acclimatized seed
sludge can efficiently remove harmful micropollutants in the influent
of moderate concentration (condition 1). Under condition 2 with ele-
vated influent concentration, the MBR with acclimatized sludge is
more effective in removing the harmful micropollutants, consistent
with Sanguanpak et al. (2019).
3.2. Bacterial community and abundance
The qPCR technique was used to quantify bacterial community in
anoxic and aerobic sludge of the experimental AS and MBR under con-
ditions 1 and 2, in terms of total bacteria and nitrifying bacteria, includ-
ing AOB, Nitrobacter, and Nitrospira.
Fig. 3 compares total bacteria, AOB, Nitrobacter, and Nitrospira of
both treatment systems under conditions 1 and 2. The bacterial commu-
nity of AS and MBR are insignificantly different (p N 0.05). Total bacteria
in anoxic and aerobic sludge of AS under both conditions are
1.67–1.71 × 1010 copies/g sludge; and AOB, Nitrobacter, and Nitrospira
are 3.43–5.60 × 105, 6.68–7.23 × 104, and 7.87–7.96 × 104 copies/g
sludge. Meanwhile, total bacteria in anoxic and aerobic sludge of MBR
under conditions 1 and 2 are 1.71–1.73 × 1010 copies/g sludge; and
AOB, Nitrobacter, and Nitrospira are 2.17–5.01 × 106, 3.21–4.28 × 105,
and 3.73–3.89 × 106 copies/g sludge.
Although total bacteria in the AS and MBR systems are insignifi-
cantly different (p N 0.05), AOB, Nitrobacter, and Nitrospira in MBR are
more abundant than in the AS system due to the presence of membrane
modules in the reactor. The small pores of the membrane modules effi-
ciently retain the sludge while discharging effluent (Sanguanpak et al.,
2019). MBR also promotes bacterial community growth (Boonnorat
et al., 2017) and retains nitrifying bacteria which assist heterotrophic
bacteria to degrade micropollutants and enhance nitrogen removal. Ni-
trifying bacteria also promote the growth of heterotrophs (Ni et al.,
2011). Given the treatment performance and micropollutant biodegra-
dation in Tables 1 and 2, the abundance and composition of bacterial

Table 2
Micropollutant concentrations of this study.

Micropollutants Unit Influent Treatment system

Two-stage AS Two-stage MBR

Anoxic Aerobic Effluent Anoxic Aerobic Effluent

BPA μg/L 51 (12)a 42 (8) 17 (9) 4 (3) 36 (11) 9 (5) ND

90 %c 100 %
P b 0.05 P b 0.05
124 (19)b 98 (12) 68 (15) 47 (14) 72 (17) 18 (16) 7 (6)
62 %d 90 %
2,6-DTBP μg/L 23 (4) 15 (3) 6 (4) 2 (3) 17 (3) 4 (2) ND
87 % 100 %
P b 0.05 P b 0.05
52 (11) 31 (8) 19 (11) 8 (5) 29 (7) 13 (5) 3 (4)
74 % 89 %
DEP μg/L 19 (7) 10 (4) 7 (3) ND 8 (6) 5 (4) ND
100 % 100 %
P N 0.05 P N 0.05
28 (5) 15 (3) 9 (4) 2 (4) 14 (4) 7 (3) 2 (5)
87 % 86 %
DBP μg/L 21 (4) 17 (7) 8 (5) 2 (3) 17 (5) 5 (3) 1 (2)
88 % 94 %
P b 0.05 P N 0.05
87 (9) 45 (8) 21 (6) 16 (4) 31 (6) 17 (4) 2 (3)
81 % 90 %
DEHP μg/L 25 (6) 16 (9) 7 (3) 3 (4) 16 (8) 5 (4) 1 (2)
81 % 93 %
P b 0.05 P N 0.05
93 (7) 68 (5) 46 (7) 32 (6) 42 (8) 22 (7) 4 (5)
65 % 87 %
DEET μg/L 19 (6) 17 (4) 8 (5) 3 (4) 17 (3) 7 (4) 2 (5)
82 % 88 %
P b 0.05 P b 0.05
24 (11) 20 (5) 13 (7) 11 (3) 17 (4) 10 (6) 3 (4)
45 % 82 %

Number of samples of each condition is 7 and values are in average (SD).

ND denotes not detected (under instrumental detection limit).
P-value of comparison between conditions 1 and 2 of the same treatment system.
a
denotes the influent concentration of condition 1 (days 0 – 42).
b
denotes the influent concentration of condition 2 (days 43 – 84).
c
denotes treatment efficiency (%) of condition 1.
d
denotes treatment efficiency (%) of condition 2.
6 A. Kanyatrakul et al. / Science of the Total Environment 724 (2020) 138275

Two-stage AS
Condition 1 Condition 2
1.0E+11
1.0E+10 Total bacteria
1.0E+09 AOB
1.0E+08
Copies/g sludge 1.0E+07 Nitrobacter
1.0E+06 Nitrospira
1.0E+05
1.0E+04
1.0E+03
1.0E+02
1.0E+01 (a)
1.0E+00
An. Ae. An. Ae.

Two-stage MBR
Condition 1 Condition 2
1.00E+11
1.00E+10 Total bacteria
1.00E+09 AOB
1.00E+08
Copies/g sludge

1.00E+07 Nitrobacter
1.00E+06 Nitrospira
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01 (b)
1.00E+00
An. Ae. An. Ae.

Fig. 3. qPCR bacterial community in bioaugmented two-stage AS and MBR systems, where An and Ae denote anoxic and aerobic respectively.

community in the two-stage MBR enhance the treatment performance
and micropollutant biodegradation. Besides, its micropollutant biodeg-
radation performance is unaffected by elevation in influent
concentrations.
3.3. Seed germination test
According to Gao et al. (2017), the phytotoxicity of xenobiotics, in-
cluding micropollutants, on plants includes morphological, physiologi-
cal, and molecular responses. Plant seed germination is crucial for
subsequent vegetative growth and development. As a result, the focus
of this research is seed germination under different influent concentra-
tions (moderate and elevated conditions) and different wastewater
treatment systems (AS and MBR with acclimatized seed sludge).
The effects of AS and MBR effluent on seed germination are deter-
mined in terms of germination rate (GR) and germination seed index
(GSI) (Aguiar et al., 2016). According to Zhang et al. (2013); Rede
et al. (2016), micropollutants adversely affect seed germination.
Table 3 compares the GR and GSI of L. sativa and V. radiata germinated
with AR and MBR effluent (of conditions 1 and 2). The two plant species
are commonly used in phytotoxicity evaluation. Under condition 1, the
GR of L. sativa and V. radiata with AS and MBR effluent are both 100%,
while the GSI of L. sativa and V. radiata with MBR effluent (1.30 and
1.54) are higher than with AS effluent (1.29 and 1.50).
Under condition 2 with elevated influent concentration, the GR of
L. sativa and V. radiata with AS effluent decrease from 100% to 80%,
and the GSI significantly decreases from 1.29 to 0.77 for L. sativa and
from 1.50 to 0.65 for V. radiata (p b 0.05). The GR with MBR effluent
remains unchanged (100%), and the GSI of L. sativa and V. radiata
(1.27 and 1.38, respectively) are insignificantly different (p N 0.05),
compared to under condition 1 (1.30 and 1.54). Meanwhile, the GR of
L. sativa and V. radiata with RO water (the control) are 100%, and the
corresponding GSI are 1.31 and 1.56. The GSI of the plant seeds germi-
nated with RO water are insignificantly different from those germinated

Table 3
Germination rate and germination seed index with two-stage AS and MBR effluent.

Plants RO water Two-stage AS effluent Two-stage MBR effluent

Control Condition 1 Condition 2 Condition 1 Condition 2

a
Lactuca sativa 100% 100% 80% 100% 100%
1.31 1.29b 0.77 1.36 1.27
P N 0.05c P b 0.05 P N 0.05 P N 0.05
P b 0.05d - P N 0.05 -
Vigna radiata 100% 100% 80% 100% 100%
1.52 1.50 0.65 1.56 1.38
P N 0.05 P b 0.05 P N 0.05 P b 0.05
P b 0.05 - P N 0.05 -
a
germination rate (GR).
b
germination seed index (GSI).
c
P-value of comparison between conditions that condition and control experiment.
d
P-value of comparison between conditions 1 and 2 of the same treatment system.
 A. Kanyatrakul et al. / Science of the Total Environment 724 (2020) 138275
with MBR effluent of both conditions (p N 0.05). This indicates that MBR
with acclimatized sludge could potentially be applied to water
reclamation.
Although the AS with acclimatized sludge could achieve high re-
moval efficiency under both conditions 1 and 2, the MBR delivers better
results since the system environment is more conducive to the growth
of effective bacterial community, as evident in the qPCR, germination
rate, and germination seed index. Fig. 4 illustrates the two-way
ANOVA of the germination rates of L. sativa and V. radiata for different
target micropollutants in AS and MBR effluent.
Miransari and Smith (2014) documented that seed germination is
subject to a number of mechanisms and factors, including plant hor-
mones. Under unfavorable conditions, plant seeds become dormant
but would germinate when the conditions are favorable. According to
Wang et al. (2015), micropollutants, including BPA, alter plant hor-
mones that regulate seed germination and plant growth.
Exposure to environmental pollutants could damage or alter plant
hormones and photosynthetic system, which in turn affects plant devel-
opment and growth (Kummerova et al., 2006; Kim et al., 2018). Accord-
ing to Gao et al. (2017), DBP and DEHP inhibit plant seed germination,
with the toxic effects most severe on root elongation and shoot
elongation.
In essence, the AS and MBR systems with acclimatized seed sludge
efficiently degrade micropollutants in the influent of moderate concen-
tration (condition 1). Under condition 2 with elevated influent concen-
tration, MBR is more effective in biodegrading the micropollutants due
to greater bacterial abundance, as evidenced by higher GR and GSI.
Therefore, MBR with acclimatized sludge is operationally and environ-
mentally more favorable, and the technology could potentially be ap-
plied to water reclamation.
4. Conclusion
This research investigates the effects of effluent from two-stage AS
and two-stage MBR systems with acclimatized seed sludge on
Fig. 4. Two-way ANOVA of the germination rates of L. sativa and V. radiata for different target micropollutants in AS and MBR effluent.
7
germination of Lactuca sativa and Vigna radiata seeds, using two concen-
trations of landfill-leachate influent: moderate (condition 1) and
elevated (condition 2). Under condition 1, the removal efficiencies for
organic compounds and nutrients (BOD, COD, TOC, TKN, NH3, TN, PO3)
of the AS and MBR are 80–96%; and 81–100% for harmful
micropollutants (BPA, 2,6-DTBP, DEP, DBP, DEHP, DEET). Under condi-
tion 2 with elevated influent concentration, MBR is more effective in
degrading micropollutants than the AS system, due to greater abun-
dance of effective bacterial groups, especially AOB, Nitrobacter, and
Nitrospira, which aid heterotrophic bacteria in biodegradation of
micropollutants and promote the growth of heterotrophs. The
micropollutant removal efficiencies of MBR are 81–100%, in comparison
with 45–87% of the AS with acclimatized seed sludge. In addition, under
condition 1, the GR and GSI of L. sativa and V. radiata germinated with AS
and MBR effluent are 100% and 1.29–1.50. Under condition 2, the GR
and GSI of plant seeds with AS effluent are reduced to 80% and
0.65–0.77, while those with MBR effluent are 100% and 1.27–1.56. The
bacterial abundance and community composition in the MBR renders
the system suitable for elevated landfill-leachate influent concentration,
as evident in higher GR and GSI. Essentially, MBR with acclimatized seed
sludge is operationally and environmentally more favorable, and the
technology holds promising potential for water reclamation.
CRediT authorship contribution statement
Alongkorn Kanyatrakul: Formal analysis, Writing - original draft.
Apichai Prakhongsak: Formal analysis, Writing - original draft. Ryo
Honda: Writing - review & editing. Supaporn Phanwilai: Formal anal-
ysis. Chairat Treesubsuntorn: Formal analysis. Jarungwit Boonnorat:
Writing - review & editing, Supervision.
Declaration of competing interest
On behalf the authors certify that they have NO interested conflict in
the subject matter or materials discussed in this manuscript.
8 A. Kanyatrakul et al. / Science of the Total Environment 724 (2020) 138275
Acknowledgments
The authors would like to express deep gratitude to the Thailand Re-
search Fund (TRF) and the Office of the Higher Education Commission
(OHEC) for research grant number MRG6180118.
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