Journal of Environmental Health Science and Engineering

https://doi.org/10.1007/s40201-020-00571-z

RESEARCH ARTICLE

Effect of sludge recirculation on removal of antibiotics in two-stage

membrane bioreactor (MBR) treating livestock wastewater
Rathborey Chan 1 & Chart Chiemchaisri 1 & Wilai Chiemchaisri 1

Received: 6 January 2020 / Accepted: 15 October 2020

# Springer Nature Switzerland AG 2020

Abstract
Two-stage MBR consisting of anaerobic and aerobic reactors was operated at total hydraulic retention time (HRT) of 48 h for the
treatment of livestock wastewater containing antibiotics, i.e. amoxicillin (AMX), tiamulin (TIA), and chlortetracycline (CTC),
under the (1st) absence and (2nd) presence of sludge recirculation between the reactors. During the operation with sludge
recirculation, the removals of organic and nitrogen were enhanced. Meanwhile, the removals of TIA and CTC were found to
decrease by 9% and 20% in the aerobic reactor but increased by 5% to 7% in the anaerobic reactor due to the relocation of
biomass from the aerobic to the anaerobic reactor. A high degree of AMX biodegradation under both anaerobic and aerobic
conditions and partial biodegradation of TIA and CTC under aerobic conditions were confirmed in batch experiments. Moreover,
the effect of sludge recirculation on biomass and pollutant removal efficiencies in the 2-stage MBR was revealed using microbial
community analyses. Membrane filtration also helped to retain the adsorbed antibiotics associated with small colloidal particles
in the system.

Keywords Antibiotics . Livestock wastewater . MBR . Sludge recirculation . SRT

Introduction the development of antibiotic-resistant pathogens. Even re-

leases of pathogenic bacteria containing integrons, capable
Improper and excessive use of antibiotics in the livestock farm of capturing and expressing arrays of genes, may conceivably
has raised concern as one of the major environmental issues accelerate the development of resistance by providing in-
worldwide [1, 2]. The residual antibiotics released together creased possibilities to probe the environmental resistance
with livestock wastes and wastewater could potentially be for novel resistance genes not yet encountered in the clinic
contaminated in the environment through intentionally [6]. Researches by Managaki et al. [7] and Hoa et al. [8]
discharging from farms with or without proper treatment, un- reported that macrolides, sulfonamides, and trimethoprim
intentional spills or release through the overflow from water were mostly found in livestock farms and some of their resid-
retention ponds in the farms during rainy seasons. The previ- ual compounds were exactly found in river water.
ous studies [3–5] investigated that the relevance antibiotics The appropriate technical solution to this problem might
used in farms were detected in different water samples, includ- depend heavily on the exposure route [9]. In current practice,
ing municipal wastewater, canal water, river water, and even stabilized pond [10], constructed wetlands [11–13], anaerobic
groundwater. Their residual compounds ultimately result in digestion [14, 15], and biological treatment system [16], given
as cost-effective and common application, have widely used
in livestock farm. Nevertheless, they were not so effective in
Electronic supplementary material The online version of this article
the removals of some residual antibiotics from livestock
(https://doi.org/10.1007/s40201-020-00571-z) contains supplementary
material, which is available to authorized users. wastewater, especially recalcitrant compounds. In general,
those systems mostly required longer biodegradation time
* Chart Chiemchaisri and thus requiring a large footprint if being designed and
[email protected] operated to achieve satisfied antibiotic removal efficiencies
which may not be possible for actual implementation in inten-
1
Department of Environmental Engineering, Faculty of Engineering, sive farming areas. Recently, the integration of advanced bio-
Kasetsart University, Bangkok 10900, Thailand logical treatment with membrane filtration, i.e. membrane
 J Environ Health Sci Engineer

bioreactor (MBR) has been developed and utilized for the 29–37). There was no sludge wastage from the 2-stage MBR
treatment of various types of wastewater. Integrated anaerobic during its operation except for sampling purpose (100 ml/
and aerobic MBR was also successfully applied in the treat- week for MLSS and residual antibiotic analyses). Initial mixed
ment of high strength wastewater containing toxic micro- liquor suspended solids (MLSS) in the aerobic reactor was set
pollutants [17]. The advantage of incorporating anaerobic at 2 g L−1. The seed sludge was obtained from the wastewater
and aerobic systems highly plays an important role in the treatment pond in the farm where livestock manure was col-
reduction of concentrated pollutants from wastewater [18]. lected, acclimatized and cultivated with feeding wastewater
Evidence of the successful application of anaerobic-aerobic under aerobic conditions before use. The livestock wastewater
system has also been confirmed in the treatment of organic was fed to the bottom of the anaerobic reactor, otherwise dur-
and nitrogen and emerging pollutants (antibiotics) from live- ing the operation with sludge recirculation, mixed with
stock farm wastewater [19]. The operation of anaerobic- recirculated sludge before entering the bottom of the anaero-
aerobic treatment in series could also be optimized for the bic reactor to allow up-flow of mixed liquor through the in-
enhancement of organic and nitrogen removals through inter- clined tube where sludge separation took place. The separated
nal recirculation [20]. It was also found that antibiotics could sludge in the anaerobic reactor was settled down and stored at
be effectively biodegraded in the aerobic system whereas an- the bottom part of the reactor while the supernatant water was
aerobic component provided complementary treatment [19]. overflown into the subsequent aerobic reactor.
In the 2-stage (anaerobic-aerobic) MBR, biomass or sludge
could be completely retained in the system through membrane Feeding wastewater and sample analyses
separation in the aerobic reactor while maintaining appropri-
ate biomass concentration through recirculation of sludge Synthetic livestock wastewater was used as feeding wastewa-
back to anaerobic reactor [21]. Therefore, the system could ter in this study. It was prepared by mixing 2 kg of wet live-
be operated without any excess sludge wastage thus providing stock manure with 100 L of tap water. After mixing well, the
unique environmental conditions for the removal of recalci- wastewater was primarily filtrated by a simple filter for reduc-
trant micropollutants [22]. Nevertheless, the effect of sludge ing a coarse particle before feeding in 2-stage MBR. The
recirculation on the performance of this 2-stage-MBR in re- characteristics of synthetic wastewater characteristics used un-
moving antibiotic contaminated in livestock wastewater is still der both operating conditions are shown in Table 1. To deter-
unclear. Therefore, this research aims at investigating the per- mine the treatment performance in terms of organic and nitro-
formance of 2-stage MBR operated under the absence and gen removals in the 2-stage MBR, influent, effluent from the
presence of sludge recirculation in removing antibiotics in anaerobic reactor and effluent from the aerobic reactor were
livestock wastewater. The treatment efficiencies of the system sampled weekly. The chemical characteristics of samples in-
and appropriate operating conditions were taken into cluded pH, suspended solids (SS), biochemical oxygen de-
consideration. mand (BOD), chemical oxygen demand (COD), total organic
carbon (TOC), ammonia nitrogen (NH3), total Kjeldahl nitro-
gen (TKN), nitrite (NO2−) and nitrate (NO3−) were analyzed in
Materials and methods triplicate following Standard Methods for the Examination of
Water and Wastewater [23]. TOC was analyzed by a TOC
Experimental setup analyzer (Shimadzu-VCSH, 511045 series). Along the opera-
tion period, the biomass concentrations in both reactors were
Laboratory scale 2-stage-MBR consisting of 30 L volume monitored in terms of mixed liquor suspended solids (MLSS)
each of anaerobic and aerobic reactors connected in series and mixed liquor volatile suspended solids (MLVSS) follow-
was used. An inclined tube (2.5 cm size) was installed in the ing the Standard Methods.
anaerobic tank for sludge separation. Four modules of sub- Three types of antibiotics such as amoxicillin (AMX),
merged hollow-fiber membrane modules (Sterapore tiamulin (TIA), and chlortetracycline (CTC) were investigated
SADF™, PVDF, 0.4 μm pore size, 0.14 m2 surface area) were in this study. The properties of antibiotic compounds are
employed in the aerobic reactor. Aeration supplied in the shown in Table A.1. Their concentrations in the influent, the
MBR tank was 15 L min−1 for maintaining dissolved oxygen effluent of the anaerobic reactor, supernatant of the mixed
(DO) in the range of 5–6 mg L−1. The permeate flux was liquor in the aerobic reactor, the effluent of the aerobic reactor,
maintained constantly at 1.25 L h−1 yielding equal hydraulic anaerobic and aerobic reactor sludge was analyzed following
retention time (HRT) of 24 h in anaerobic and aerobic reactors standard extraction procedures [24] using Oasis HLB 200 mg,
(total HRT of 48 h). The 2-stage MBR was operated under 6 mL (Waters, MA, USA.). The concentrations of antibiotics
two different conditions i.e. 1st condition: no sludge recircu- in samples and standards were determined by Shimadzu High-
lation (week 1–28) and 2nd condition: with sludge recircula- Performance Liquid Chromatograph (HPLC) equipped with
tion at a rate of 100% of wastewater feeding flow rate (week an ultraviolet (UV) detector. The type of column detection
J Environ Health Sci Engineer

Table 1 Characteristics of synthetic wastewater and removal performance of 2-stage MBR

Parameter 1st Condition (No sludge recirculation) * 2nd Condition (100% sludge recirculation) **

Influent Anaerobic effluent Aerobic effluent %R Influent Anaerobic effluent Aerobic effluent %R

pH 7(0.3) 7.1(0.2) 6.7(0.5) – 7.7(0.2) 7.4(0.2) 7.4(0.2) –

SS (mg/L) 485.2(252.4) 242.4(170.4) 4(3.1) 98.6 553.5(329.9) 225.8(121.6) 1.0(0.7) 99.6
BOD (mg/L) 1127.1(410.1) 483.6(217.6) 14.7(17.2) 98.5 940.9(436.7) 226.2(206) 6.5(5) 99.2
COD (mg/L) 2151.8(720.9) 986.4(427.9) 117.9(43.3) 93.5 1921.2(676.8) 759.7(562.7) 88(27.6) 95.5
TOC (mg/L) 290.4(162.3) 121.9(60.9) 36.1(15.8) 84.6 200.0(71.9) 65.8(19) 26.1(10.9) 84.4
NH3-N (mg/L) 105.2(37.8) 89.6(40.1) 7(4.4) 93.5 90.0(20.4) 71.2(19.3) 1.2(1.1) 98.1
TKN (mg/L) 152.6(41.0) 124.5(35.2) 12.3(9.8) 91.8 152.3(36.4) 117.8(74) 2.1(1.6) 96.5
NO2-N (mg/L) 0.5(0.2) 0.4(0.1) 2.2(1.6) – 0.5(0.2) 0.3(0.1) 1.0(0.6) –
NO3-N (mg/L) 0.9(0.5) 1.1(0.7) 1.4(0.9) – 0.5(0.1) 0.6(0.1) 1.9(0.6) –

* No. of samples = 28 (week 1–28), ** No. of samples = 8 (week 29–37)

was Intertsil® ODS-3 (5um 4.6 × 250 mm). The detection while a control experiment conducted using synthetic waste-
limit of the analyzed was 0.1 μg L−1. For AMX analyses, water and antibiotic compounds without the presence of
the mobile phase consists of a 95% phosphate buffer sludge were also performed. To distinguish the removals
(0.01 mol L−1), pH = 5.5 and 5% of acetonitrile mixture. The through adsorption and biodegradation, parallel batch experi-
mobile phase flow rate of 1.3 ml/min and UV detection at ments under the same conditions as above were conducted
200 nm were used. The detection of TIA was carried out by using anaerobic and aerobic sludge samples autoclaved under
using a mobile phase of an 87% phosphate buffer 121 °C for 15 min thrice to quantify the amount of antibiotics
(0.025 mol L−1), pH = 2.5 and 13% of acetonitrile mixture. removed through adsorption on the sludge particles. Each
Similar conditions to AMX of mobile phase flow rate and batch experiment was performed over 12 h during which rep-
UV detection was also used. For CTC detection, the mobile resentative samples were collected at every 2 h interval. The
phase consists of a 65% oxalic acid sodium salt differences in antibiotic removals using active and inactive
(0.01 mol L−1), 25% of acetonitrile and 10% methanol mix- sludge were considered as the removals through biodegrada-
ture. This antibiotic was detected under a mobile phase flow tion. Moreover, the biodegradation rate constants of studied
rate of 1 ml/min and UV detection of 250 nm. The temperature antibiotics by anaerobic and aerobic sludge under different
of the detector used for all targeted antibiotics was 40 °C. conditions were derived following the first-order kinetic as
explained in Kimura et al. [25] and Abegglen et al. [26].

Study of antibiotic removals in batch experiments

Microbial community analyses
To clarify the antibiotic removal mechanism in anaerobic and
aerobic reactors, batch experiments to determine the removals The microbial sludge samples (50 ml) from anaerobic and
of antibiotics through adsorption and biodegradation by mi- aerobic reactors operated under the 1st condition (at week
crobial sludge obtained from the reactors during the steady 22) and the 2nd condition (at week 38) were collected and
operation were carried out. The batch experiment was per- analyzed for the microbial communities using next generation
formed in a serum bottle with a sample volume of 100 mL. sequencing technique. For these analyses, the isolation of the
The serum bottle was covered by aluminum foil to prevent microbial genomic DNA from the test materials were per-
possible photo-degradation and other contaminations. For an- formed by the DNeasy PowerSoil Pro Kit. The 16S rRNA
aerobic reactor sludge testing, different sludge concentrations gene was amplified using 341F and 805R primers (Quanta
of 1, 5 and 10 g L−1 were used in batch experiments to inves- bio, USA). The bioinformatics of relative abundances (%) of
tigate the effect of sludge concentration during its built-up in bacteria or archaea from each sample was defined and
the reactor on the antibiotic removal capacities. Meanwhile, demultiplexed by using Miseq Reporter Software V3.1.
aerobic reactor sludge cultivated under the absence and pres-
ence of sludge recirculation was investigated in batch experi- Statistical analysis
ments under aerobic conditions. The sludge concentration was
set at 2 g L−1 under both conditions. The initial antibiotic Microsoft Excel version 2010 was used to perform statistical
concentrations used in all batch experiments were set at analyses of the experimental data. The significant difference
1000 μg L−1. All the experiments were carried out in duplicate (p < 0.05 in one-way ANOVA analysis) between the water
 J Environ Health Sci Engineer

quality parameters, antibiotic concentrations, and removal the anaerobic reactor by increasing its biomass concentration.
performance of antibiotics between the 1st and 2nd conditions Meanwhile, the mass of TKN removed in the aerobic reactor
of the 2-stage MBR operation was analyzed. was maintained at the same level as the 1st condition thus
resulting in the low level of NH3 and TKN in the aerobic
reactor effluent (1–2 mg L−1).
Results and discussion During the operation period, there was no significant in-
crease in the transmembrane pressure of hollow-fiber mem-
Treatment performance of the 2-stage MBR brane modules observed. The solid particles incoming with
the livestock wastewater were partially removed in the anaer-
The performance of 2-stage MBR operated under the absence obic reactor by about 50% and their remaining was almost
and presence of sludge recirculation is presented in Table 1. completely removed by submerged membrane modules in
During the operation without sludge recirculation (1st condi- the aerobic reactor resulting in total removals of about 99%.
tion), the biomass (MLSS) concentrations in the aerobic reac- Meanwhile, most of the MLSS increase in the reactors would
tor were gradually increasing from 2 g L−1 to 5–6 g L−1 after be a result of microbial cell growth on organic substrates. In
12 weeks of operation. The average and maximum biomass previous research [27], no significant effect of sludge concen-
increasing rates were found to be 0.05 and 0.14 g L−1 d−1 tration in MBR on membrane fouling was observed when the
respectively. Afterward, the biomass concentrations remained MBR was operated at MLSS of less 10 g L−1 corresponding to
relatively constant in a narrow range between 5.1–6.4 g L−1 the mixed liquor viscosity between 5 to 10 N m−2 s−1.
(5.9 g L−1 on average). Simultaneously, anaerobic sludge con- Moreover, the high aeration rate (15 L min−1) in the MBR
centrations in the first reactor were gradually increased from system could reduce the possibility of membrane fouling
2 g L−1 to about 10 g L−1. The biomass in both reactors [28] and constant permeate (30 L d−1) could be maintained.
contained high volatile solids content, i.e. MLVSS/MLSS of In terms of the treatment performance, there was no signif-
0.80–0.85. During this operation period, average organic re- icant difference in the overall treatment performance in terms
moval in terms of BOD, COD and TOC removal efficiencies of organic but nitrogen removals in the 2-stage MBR operated
were 98.5%, 93.5%, and 84.6% in which about 60% of organ- under 1st and 2nd conditions (p value >0.05). Moreover, there
ic removal took place in the anaerobic reactor. Meanwhile, is some notable difference in the contribution from the anaer-
average NH3-N and TKN removals of 93.5% and 91.8% were obic and aerobic reactors in the overall treatment efficiencies.
achieved. It was noticed that, along the operation period, re- The BOD and COD removals in the anaerobic reactor were
maining NH3 and TKN concentrations after the aerobic treat- found to be about 60% under the 1st condition but they were
ment was increasing which indicated that the nitrification re- improved to 67–70% in the 2nd condition. In terms of nitro-
actor could be partially inhibited during the biomass built-up gen (NH3 and TKN) removals, there were also improved from
in the reactor. Simultaneously, the oxidized nitrogen could be 15% and 18% under the 1st condition to 20% and 22% under
denitrified in this aerobic reactor operated under high biomass the 2nd condition. As described earlier, a notable enhance-
concentrations, thus, yielding their remaining concentrations ment of the nitrification when the aerobic reactor of 2-stage
of about 1–2 mg L−1 in the effluent. MBR was operated under lower biomass concentration from
When sludge recirculation was introduced in the 29th sludge recirculation operation. This operation helped to pro-
week, there was a drastic decrease in biomass concentrations mote simultaneous nitrification and denitrification reactions
in the aerobic reactor by about 70%. As a result, the MLSS responsible for the majority of nitrogen removals in the aero-
concentrations were reduced to 1.6–2 g L−1 at the 32nd week. bic reactor while recirculating back some remaining oxidized
Meanwhile, the sludge concentrations in the anaerobic reactor nitrogen back to the first reactor where further denitrification
were increasing from 10 to 37 g L−1 from this recirculation could proceed. The promotion of denitrification in the first
practice. Despite having lower biomass concentrations main- reactor also helped to improve organic carbon removal
tained in the aerobic reactor under this operation, high organic through their use as an electron donor.
removals of 99.2% for BOD, 95.5% for COD and 84.4% of
TOC could be achieved. On the other hand, this operation at Antibiotic removals in the 2-stage MBR
lower biomass concentration helped to enhance oxygen trans-
fer for the microbial activities and thus improved NH3 and Table 2 demonstrated the average concentrations of targeted
TKN removals through nitrification to 98.1% and 96.5%. antibiotics (AMX, TIA, and CTC) and their removals in the 2-
This enhancement of oxygen availability was evidenced by stage MBR operation. During the operation, the concentra-
an increase in dissolved oxygen (DO) concentration measured tions of antibiotics varied depend on their residual amount
at 5 to 6.5 mg L−1 in the aerobic reactor under 1st and 2nd contained in the livestock manure. During the 1st condition,
conditions respectively. An operation under sludge recircula- antibiotic removals were started to be monitored on the 24th
tion condition slightly improved NH3 and TKN removals in week. Overall, it was found that the averaged antibiotic
J Environ Health Sci Engineer

Table 2 Antibiotic concentrations and their removal efficiencies in the 2-stage MBR

Antibiotic Influent Anaerobic effluent Aerobic effluent % Removal

μg/L
Max Mean Min Max Mean Min Max Mean Min Anaerobic Aerobic

1st condition: no sludge recirculation

AMX 2104.5 576.4 94.1 985.5 219.4 14.3 2.1 0.7 0.0 77.6 98.8
TIA 2224.8 1041.6 187.4 1066.6 516.1 106.7 211.7 77.0 1.6 47.2 83.4*
CTC 5888.0 1616.7 229.7 3187.1 880.7 47.2 9.2 2.2 0.0 45.9 97.6*
2nd condition: 100% of sludge recirculation
AMX 180.6 89.2 5.2 83.5 18.9 0.0 4.0 0.5 0.0 76.0 98.0
TIA 397.8 233.8 123.0 250.9 114.9 35.8 33.8 23.5 16.7 53.6 74.3*
CTC 6339.4 2285.4 75.6 3258.3 1209.2 3.7 639.4 276.5 0.0 51.1 74.2*

*Significant difference between the operating conditions (p < 0.05)

removal efficiencies in the anaerobic and aerobic reactors value of Log P, i.e. 0.87 suggesting its hydrophilic property.
were 56.9%, 93.3% and 60.2%, 82.1% during the 1st and In the 2-stage MBR, low ratios of anaerobic effluent to influ-
2nd conditions respectively. During the operation under the ent (A/I), supernatant to anaerobic effluent (S/A), and mem-
1st condition, the removals of AMX, TIA, and CTC in the brane permeate to the supernatant (P/S) of AMX was ob-
anaerobic and aerobic reactors were 77.6%, 47.2%, 45.9%, served during its treatment (Fig. 1). Therefore, the AMX ex-
and 98.8%, 83.4%, 97.6%, respectively. Meanwhile, they hibited high removal in the liquid phase of anaerobic and
were 76%, 53.6%, 51.1% and 98%, 74.3%, 74.2% under the aerobic reactors and very low residual concentrations were
2nd condition. There was much difference in AMX concen- found associated with solid particles in those reactors.
trations in the influent and supernatant of anaerobic reactor Figure 2 demonstrates that 69% and 77% AMX removal in
between the 1st and 2nd conditions but no significant differ- the anaerobic and aerobic reactors under the both conditions
ence in AMX removals between both conditions was ob- came from its biodegradation while adsorptive removal was
served (p > 0.05). Nevertheless, average TIA and CTC re- less than 1%. However, its biodegradation under 2nd condi-
movals were found insignificantly increased by 5% to 7% in tion was reduced by about 5% due the reduction of active
the anaerobic reactor (p > 0.05) but significantly decreased by microorganisms from biomass recirculation whereas mem-
9% to 20% in the aerobic reactor under the 2nd condition brane filtration could retain residual AMX up to 10%.
(p < 0.05). The increase of sludge concentrations from Zuccato et al. [34] also confirmed susceptible to AMX to
10 g L−1 to 37 g L−1 in the anaerobic reactor and the decrease microbial degradation and hence its removal efficiencies were
of biomass in the aerobic reactor from 6 g L−1 to 2 g L−1 when found higher than 99%. Recent research by Perini et al. [35]
sludge recirculation was introduced affected antibiotic remov- also reported that AMX could be completely biodegraded
al performance of the 2-stage MBR. The antibiotics were re- under anaerobic condition yielding effluent concentrations
moved via biotic (biodegradation, mainly by active microor- lower than the detection limit (<0.1 μg L−1). Overall, AMX
ganisms in the reactor) and abiotic (e.g. sorption to colloidal mainly removed in this 2-stage MBR through biodegradation.
particles, hydrolysis) mechanisms [29]. Therefore, the shift of For more hydrophobic compound, i.e. TIA (log P = 4.75),
sludge concentrations in both reactors from sludge recircula- its removal efficiencies were found much lower than those of
tion would directly influence the biodegradation and adsorp- AMX both under the absence or presence of sludge recircula-
tion rates of antibiotics [30]. tion. However, an introduction of sludge recirculation slightly
Otherwise, the removal mechanism of AMX in the anaer- increased in the removal in the anaerobic reactor by 7% but
obic and aerobic system may be mainly occurred by biodeg- decreased the removal in the aerobic reactor by 9%. The main
radation following initial adsorption [31, 32]. In fact, β- removal pathway of TIA was adsorption followed by partial
lactams are all related structurally though the presence of a biodegradation. In the previous study, TIA was reported as the
β-lactam ring, which is readily degraded through hydrolytic persistent compound in the environment and no biodegrada-
cleavage and ultimately mineralized to CO2 and water [33]. tion was detected even after 180 days incubation [36]. From
Moreover, high transformation and removal of AMX may the aerobic biodegradability test in previous research [37], no
also due to the structural similarity of AMX to other natural degradation of TIA was observed until the 20th day and
substances, which render to microorganisms. Likewise, high reached only 40% after 43 days, while abiotic degradation
biodegradability of AMX was found associated with a low has not been detected (≤ 2%). Another study reported the
 J Environ Health Sci Engineer

Fig. 1 Concentration ratio of

anaerobic effluent to influent
(A/I), supernatant in aerobic
reactor to anaerobic effluent
(S/A), membrane permeate to
aerobic reactor supernatant (P/S)
and accumulation in anaerobic
(SAN) and aerobic (biomass)
sludge

ability of wood-rot fungal strains to eliminate 10 mg L−1 TIA Bosevski et al. [37] reported that 600 mg L−1 of TIA in waste-
from 65 to 95% over a period of 12 days by manganese per- water did not affect heterotrophic activities but inhibited nitri-
oxidase [38]. Nevertheless, at least 5%–15% of TIA was fication. In this study, it was found that nitrification and nitro-
found removed via biodegradation in this study. Ben et al. gen removal was enhanced during the operation with sludge
[39] reported that the biological treatment system could not recirculation (2nd condition), therefore, residual TIA in the
complete the removal of TIA via biodegradation therefore aerobic reactor at the level of few mg L−1 did not provide
advanced oxidation, i.e. ozonation was utilized as a polishing any adverse impact on nitrification.
process for further treatment. The observation of high A/I of For CTC, there was a significant difference in their ob-
0.6 and 0.46 of TIA under the 1st and 2nd conditions, high served removal efficiencies during the 1st and 2nd conditions.
removal of TIA in the anaerobic reactor took place in parallel Under the operation with sludge recirculation, the removal in
with increasing sludge concentration. From the above obser- the anaerobic reactor was increased by about 7% but decrease
vation, TIA removal in anaerobic reactor may be mainly by 24% in the aerobic reactor. The presence of sludge recir-
caused by adsorption because its adsorptive capacity to anaer- culation also resulted in a significant increase in its accumu-
obic sludge under the 2nd condition was observed to increase lation in anaerobic and aerobic sludge (Fig. 1), showing resid-
around 10% as compared to the 1st condition. Moreover, the ual concentration of TIA in anaerobic sludge and biomass up
removal efficiencies in the aerobic reactor under both condi- to 0.25 μg g−1. This account for almost 2.5 times which is
tions were found lower than 20% while membrane filtration higher than residual CTC in anaerobic sludge and biomass
could retain residual TIA up to 30%. The distribution of TIA under 1st condition. CTC is one of the tetracycline com-
in solid and liquid forms (Fig. 2) also exhibited low biodeg- pounds, which was demonstrated as complex properties and
radation of TIA in the 2-stage MBR. Overall, highly concen- easily bind to calcium and similar ion thus it tends to be stable
trated anaerobic sludge maintained under the 2nd condition compounds and further binds to colloidal particles and sludge
may enhance the adsorption of TIA in sludge and fine [40]. The sorption tendency of CTC may originate from elec-
suspended particles while some removals occurred through trostatic forces rather than hydrophobic interactions and sorp-
biodegradation and membrane filtration in the aerobic reactor. tion of these compounds is likely due to fast mass transfer of

Fig. 2 Fate of antibiotic

compounds observed in the 2-
stage MBR, i.e. total biodegrada-
tion (T.Bio), effluent, biomass,
supernatant MBR (S.MBR),
sludge anaerobic (S.Ana) effluent
anaerobic (Eff.Ana)
J Environ Health Sci Engineer

solute from the liquid phase to the boundary layer of the MBR of sludge decreases the amount of those active microorgan-
sludge [41]. A notable change in A/I, S/A, and P/S ratio be- isms in the aerobic reactor. Moreover, it was reported that the
tween the 1st and 2nd conditions suggested that there was a biodegradation of TC compounds became slower when their
much-reduced capability for CTC elimination in the system. concentration was elevated [43]. Meanwhile, Chen et al. [44]
Meanwhile, membrane filtration played a significant role in reported that the presence of CTC at 1–10 mg L−1 level pro-
retaining the untreated compound in the aerobic reactor under vided a major impact on the denitrification process but the
both operating conditions. There was a reasonable CTC bio- only marginal effect on nitrification. In this study, the pres-
degradation in the 2-stage MBR under the 1st condition ence of CTC was found up to 6 mg L−1 and but no adverse
(Fig. 3), showing the distribution percentage up to 45%. But effect on nitrification and denitrification in the aerobic reactor
it was substantially reduced when sludge recirculation was was noticed.
introduced under the 2nd condition. Therefore, an introduc-
tion of sludge recirculation possibly reduced the retention of Antibiotic removal mechanisms
CTC degrading microorganisms, especially in the aerobic re-
actor. Generally, a high SRT condition similar to this 2-stage The removal mechanisms of investigated antibiotics were ver-
MBR could allow the enrichment of slowly growing bacteria ified in batch experiments. Figures 3 and 4 show the AMX,
and provides a greater diversity of enzymes capable of degra- TIA and CTC concentration profiles along the time course of
dation antibiotic compounds [42]. However, the recirculation experiment removal rates using abiotic and biotic sludge

Fig. 3 Removal of antibiotics through adsorption and biodegradation by (S.Ana 1 g L−1), e TIA (S.Ana 5 g L−1), f TIA (S.Ana 10 g L−1), g CTC
anaerobic sludge (S.Ana) under different concentrations, a AMX (S.Ana (S.Ana 1 g L−1), h CTC (S.Ana 5 g L−1), i CTC (S.Ana 10 g L)
1 g L−1), b AMX (S.Ana 5 g L−1), c AMX (S.Ana 10 g L−1), d TIA
 J Environ Health Sci Engineer

Fig. 4 Removals of antibiotics through adsorption and biodegradation by aerobic reactor sludge cultivated under different conditions; a AMX 1st
condition, b AMX 2nd condition, c TIA 1st condition, d TIA 2nd condition, e CTC 1st condition, f CTC 2nd condition
J Environ Health Sci Engineer

under anaerobic and aerobic conditions. The difference in the was low (2.9 × 10−4-4.5 × 10−4 h−1) as presented in Table 3.
mass removal between those two sludge conditions was used The high adsorption capacity of TIA could be explained by its
in the derivation of first-order reaction rate constant for anti- low water solubility property expressed in terms of log P [45].
biotic removals through biodegradation of the 2-stage MBR The high value of log P (>4.0) demonstrates a high capacity of
sludge as presented in Table 3. antibiotics to adsorb on sludge and colloidal particle through
For AMX, the results have clearly shown that biodegrada- its high hydrophobicity. Under the aerobic condition, adsorp-
tion was the main mechanism responsible for its removal. tion of TIA predominated its removal during the initial period
Under anaerobic conditions, the amount of AMX adsorbed (up to 4 h) while biodegradation further proceeded later on
and biodegraded varied with the sludge concentrations (Fig. (Fig. 4c-d). When there was no biodegradation activity in the
3a-c). The first-order rate constant for its biodegradation in- system, the desorption of TIA occurred. Thus, most residual
creased from 1.2 × 10−1 h−1 at 1 g L−1 to 1.9 × 10−1 h−1 at concentrations TIA were contained in the supernatant and
10 g L−1. Due to the quick reaction of AMX to microbial suspended particles. A higher biodegradation rate of TIA in
degradation [34], a higher concentration of anaerobic sludge the aerobic condition was found under the absence of sludge
incubation led to a higher biodegradation rate. Under the aer- recirculation (4.1 × 10 −2 h −1 ) as compared to that of
obic condition, there was only a slight difference in the re- recirculated sludge (2.3 × 10−2 h−1). These values were found
movals of AMX using aerobic reactor sludge cultivated under much lower than those of AMX. Bosevski et al. [37] found
the absence and presence of sludge recirculation. It was found only 20% biodegradation of TIA during 41 days period with a
that the recirculated sludge provided slightly higher AMX low degree of abiotic removal (<2%). Some degree of TIA
adsorption during the initial period of batch experiments biodegradation observed under the aerobic condition in this
(Fig. 4a-b) thus yielding lower biodegradation rate constant study could contribute from the diverse enzyme produced
(4.0 × 10−1 h−1) compared to that of non-recirculated sludge from biomass cultivated in the MBR operated under long
(6.6 × 10−1 h−1). The biodegradation rates of AMX under aer- sludge age condition [22]. Nguyen et al. [46] also reported
obic conditions were about 2–3 times of those under anaerobic high removal of TIA of 2.5–200 mg/L at 60.1–99.9% in
condition. These results would suggest that there was no sig- 16 h using enriched microbial consortium cultivated from
nificant difference in the removals of AMX operated under the livestock wastewater. A low concentration of TIA (1 mg/L)
absence or presence of sludge recirculation as their biodegra- employed in the batch experiment did not create a toxic con-
dation capabilities would be sufficiently maintained. dition to nitrifying bacteria, which also supported the biodeg-
Nevertheless, the adsorptive removal of AMX in the anaero- radation of recalcitrant organic compounds through co-
bic reactor would increase with time when biomass was accu- metabolism [47].
mulated in the reactor under the operation without sludge Likewise, the removals of CTC under anaerobic condition
wastage from the system. Subsequently, the adsorbed AMX was also mainly from adsorption at about 60% regardless of
would be further removed through biodegradation in the an- sludge concentrations (Fig. 3g-i). Low values of biodegrada-
aerobic and aerobic reactor thus there was no significant ac- tion rates (2.5 × 10−3-4.3 × 10−3 h−1) were also derived
cumulation of AMX observed during the operation of the 2- (Table 3). In contrast to AMX, CTC with low log P value
stage MBR system. (0.11) was found partially adsorb in sludge and colloidal par-
Meanwhile, TIA was mainly removed through adsorption ticles. It is anticipated that the tetracycline (TCs) compound
onto sludge particles. Under anaerobic condition, the adsorp- containing high molecular weight and complex structure dem-
tion capacity of TIA was found independent of the sludge onstrate more resistant to biotransformation than those with
concentrations used (Fig. 3d-f). Nevertheless, TIA desorption low molecular weight [48]. However, reasonable biodegrada-
from sludge particles was observed and it was affected by the tion of CTC was observed in the aerobic system under both 1st
sludge concentration. The biodegradation rate constant of TIA and 2nd conditions (Fig. 4e-f, Table 3). Partial reduction of

Table 3 First order rate constant (Kbio) of antibiotic biodegradation by anaerobic and aerobic reactor sludge

Compound First-order rate constant, Kbio (h−1)

Anaerobic reactor sludge Aerobic reactor sludge (2 g L−1)

1 g L−1 5 g L−1 10 g L−1 1st condition 2nd condition

AMX 1.2 × 10−1 1.3 × 10−1 1.9 × 10−1 6.6 × 10−1 4.0 × 10−1
TIA 4.5 × 10−4 4.0 × 10−4 2.9 × 10−4 4.1 × 10−2 2.3 × 10−2
CTC 4.3 × 10−3 4.1 × 10−3 2.5 × 10−3 5.0 × 10−2 2.0 × 10−2
 J Environ Health Sci Engineer

biodegradation activity may relate to the loss of specific mi- the complete oxidizing ammonium to nitrogen by using oxi-
croorganisms from the aerobic reactor through sludge recircu- dized nitrogen as electron acceptor [53–55].
lation. Chang et al. [43] found that the degradation of CTC After sludge recirculation was introduced, there was a sig-
was reflected by 10 bacterial strains capable of aerobically nificant change of microbial communities in the anaerobic
degradation of CTC as a carbon source. Moreover, the re- reactor. The relative abundance of Firmicutes (Clostridia
placement of sludge from the anaerobic system to the aerobic class) decreased to 38.4%, while Synergistetes and
system may not be effective in the biodegradation of recalci- Euryrchaeota were ranged from 0.03% to 0.06%, respectively.
trant compounds like CTC and TIA. The reduction of those bacteria in anaerobic sludge might be
Considering the removals of these studied antibiotics to caused by intrusion of dissolved oxygen together with
their chemical properties, it confirmed that the antibiotic with recirculated sludge, which could inhibit the growth of anaer-
hydrophilic property (AMX) was mainly biodegraded in the obic fermentation bacteria. But other phyla such as
2-stage MBR whereas those with hydrophobic properties Actinobacteria (7% Actinobacteria class), Chloroflexi (8.5%
(TIA, CTC) were mainly removed through adsorption onto Chloroflexia and 6% Anaerolineae classes), Planctomycetes
sludge and small colloidal particles. They can also be present- (3.2% Planctomycetacia and 4.2% Phycisphaerae classes),
ed in the supernatant of anaerobic and aerobic reactors. The Proteobacteria (3.1% Alphaproteobacteria class) and
biotransformation of adsorbed compounds in sludge particles Gemmatimonadetes (7.5% Gemmatimonadetes class) were
would occur either yielding further reduction via microbial highly detected in anaerobic sludge. Likewise, most those
degradation or desorption from solid particles, if not bacteria including Firmicutes (16.2% Clostridia class) were
biodegraded, once their maximum adsorption capacity onto also the predominant bacteria in the aerobic reactor, showing
sludge particles has been reached [49]. In this study, small relative abundance ranging from 2.3% to 24.9%. Nitrospira
percentages of their removals were observed in the anaerobic and Alphaproteobacteria classes were decreased to 1.7% and
reactor, but the removals took place to a greater extent in the 4% while Anaerolineae class was increased to 14%, accord-
aerobic reactor. ingly. Based on the results above, an observation of low level
of oxidized nitrogen in the aerobic reactor under this condition
Microbial communities could be clarified by the co-existence of nitrifying and
denitrifying bacteria in the reactor.
The microbial communities in anaerobic and aerobic reactors For antibiotics removal, the presence of microorganisms
under the 1st and the 2nd conditions are presented in Fig. 5. responsible for their biodegradation in both reactors could
The microbial analyses revealed the presence of microorgan- explain high degree of AMX observed in the 2-stage MBR.
isms classified in 15 phyla, 88 classes, 93 families and 154 Yang et al. [56] reported the presence of AMX-degrading
genera in total. bacteria in pig manure which grew at high growth rate
In the 1st condition, Firmicutes (88%) was predominantly (68%) under optimum condition (pH of 6.9, 35 °C) during
in the anaerobic reactor following by Synergistetes (3.8%), 50 h fermentation. For TIA degradation, recent study by
Euryarchaeota (2.8%), and Actinobacteria (2.1%). Those phy- Nguyen et., al [46] suggested the major role of phylum
la contain Clostridia, Synergistia, Methanobacteria and Proteobacteria belonging to genera Achromobacter, Delftia,
Methanomicrobia classes. They are anaerobic bacteria com- Flavobacterium, Pseudomonas, and Stenotrophomonas in
monly involve in anaerobic methane fermentation processes degrading TIA with high efficiencies (60.1–99.9%) within
[50, 51]. Meanwhile, Actinobacteria (Actinobacteria class) 16 h. Even though those genera were not detected in this
could reduce nitrate, nitrite, and nitric oxide to nitrous oxide study, high relative abundance of the phylum Proteobacteria
under anaerobic conditions [52]. In the aerobic reactor, the (18.2%) in aerobic sludge under the 1st condition could pos-
most predominant phylum was Planctomycetes (23.7% sibly yield high TIA degradation. In the 2nd condition, TIA
Planctomycetacia class) following by Proteobacteria (18.2% degradation drastically decreased in the aerobic reactor togeth-
Alphaproteobacteria class), Nitrospira (18.4% Nitrospira er with a decrease in relative abundance of Proteobacteria to
class), Firmicutes (13.9% Clostridia class), Chloroflexi 4%. Meanwhile, its degradation may slightly increase in the
(5.5% Chloroflexia class), and Actinobacteria (3.4% anaerobic reactor due to the increase of Proteobacteria. In case
Actinobacteria class). The presence of predominant bacteria of CTC, previous report by Chang et al. [43] demonstrated
in suggested the possibility of having anoxic zone in the aero- that CTC degradation was highly achieved by 10 aerobic bac-
bic reactor under high biomass condition, which is suitable for terial strains whereas Aydin et al. [57] also reported that some
anaerobic denitrification process in the aerobic reactor. Thus, denitrifying bacteria could also reduce nitrate using CTC as
high nitrogen removal could be achieved because the genera organic substrate for assimilation and energy generation in
levels of Amaricoccus, Nitrospira and Uncultured Anaerobic case of the absence of organic electron source. From such
Ammonium Oxidizing Bacterium in Alphaproteobacteria, information, the decrease CTC degradation in the aerobic re-
Nitrospira and Chloroflexia classes could be responsible for actor under the 2nd condition could be related to the decrease
J Environ Health Sci Engineer

Fig. 5 Microbial community

structure at phylum and class in
anaerobic and aerobic MBR
sludges under 1st and 2nd
conditions
 J Environ Health Sci Engineer

of aerobic microbial population. However, the presence of surface water of a livestock production region in northern China.
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24 h could provide high organic and nitrogen removals drug resistant integron-bearing bacteria. PLoS One. 2013;8(10):
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AMX, TIA, and CTC, was removed in a 2-stage MBR system Distribution of macrolides, sulfonamides, and trimethoprim in trop-
through different mechanisms. AMX found highly ical waters: ubiquitous occurrence of veterinary antibiotics in the
biodegraded in both anaerobic and aerobic reactors while Mekong delta. Environ Sci Technol. 2007;41:8004–10.
TIA and CTC were partially biodegraded under aerobic con- 8. Hoa PTP, Managaki S, Nakada N, Takada H, Shimizu A, Anh DH,
et al. Antibiotic contamination and occurrence of antibiotic-
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the followed by the order, AMX > CTC > TIA. An operation Sci Total Environ. 2011;409(15):2894–901.
of 2-stage MBR at HRT of 24 h could not completely remove 9. Larsson DGJ. Antibiotics in the environment. Ups J Med Sci.
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10. de Godos I, Muñoz R, Guieysse B. Tetracycline removal during
degradation but an increase in sludge accumulation in the
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the compounds within the system as long as the maximum 11. Liu L, Liu C, Zheng J, Huang X, Wang Z, Liu Y, et al. Elimination
adsorption capacities have not been reached. After introduc- of veterinary antibiotics and antibiotic resistance genes from swine
wastewater in the vertical flow constructed wetlands.
tion of sludge recirculation, there was no significant impact in
Chemosphere. 2013;91(8):1088–93.
AMX degradation. In contrast, TIA and CTC degradation 12. Huang X, Liu C, Li K, Su J, Zhu G, Liu L. Performance of vertical
extremely reduced in aerobic reactor due to the reduction rel- up-flow constructed wetlands on swine wastewater containing tet-
ative abundance of their active microorganisms i.e., racyclines and tet genes. Water Res. 2015;70:109–17.
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Proteobacteria and Nitrospira. However, membrane filtration
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tions within the system. substrate type. Chem Eng J. 2017;308:692–9.
14. Widyasari-Mehta A, Suwito HRKA, Kreuzig R. Laboratory testing
Funding The research work was carried out as an activity in the 20th on the removal of the veterinary antibiotic doxycycline during long-
Anniversary Commemorative Research Project of Kurita Water and term liquid pig manure and digestate storage. Chemosphere.
Environment Foundation (KWEF). The authors also received a collabo- 2016;149:154–60.
rative research (CR) fund from ASEAN University Network-Southeast 15. Feng L, Casas ME, Ottosen LDM, Møller HB, Bester K. Removal
Asia Engineering Education Development Network (AUN/SEED-Net) of antibiotics during the anaerobic digestion of pig manure. Sci
supported by Japan International Cooperation Agency (JICA) to carry Total Environ. 2017;603:219–25.
out a preliminary investigation. 16. Batt AL, Kim S, Aga DS. Comparison of the occurrence of antibi-
otics in four full-scale wastewater treatment plants with varying
designs and operations. Chemosphere. 2007;68(3):428–35.
Compliance with ethical standards 17. Kaewmanee A, Chiemchaisri W, Chiemchaisri C. Influence of high
doses of antibiotics on anoxic-aerobic membrane bioreactor in
Conflict of interest The authors declare that they have no competing treating solid waste leachate. Int Biodeterior Biodegrad.
interests. 2019;138:15–22.
18. Sandra A, Jamil NA, Jabbar S, Sakyat S, Gomes C. Aerobic and
anaerobic sewage biodegradable processes: the gap analysis. Int J
Res Environ Sci. 2017;3(3):9–19.
19. Chen J, Liu YS, Zhang JN, Yang YQ, Hu LX, Yang YY, et al.
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