Understanding the role of extracellular polymeric substances (EPS) on

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Understanding the role of extracellular polymeric substances (EPS) on ciprofloxacin (CIP) adsorption in aerobic sludge, anaerobic sludge and sulfate-reducing bacteria (SRB) sludge systems Huiqun Zhang, Yanyan Jia, Samir Kumar Khanal, Hui LU, Heting Fang, and Qing Zhao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00568 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Environmental Science & Technology

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Understanding the role of extracellular polymeric substances (EPS)

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on ciprofloxacin (CIP) adsorption in aerobic sludge, anaerobic sludge

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and sulfate-reducing bacteria (SRB) sludge systems

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Huiqun Zhang1,2#, Yanyan Jia1,2#, Samir Kumar Khanal3, Hui Lu*1,2, Heting

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Fang1,2, Qing Zhao4

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1

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China

School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, 510275,

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2

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Technology (Sun Yat-sen University), Guangzhou, 510275, China

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3

Department of Molecular Biosciences and Bioengineering, University of Hawai`i at Mānoa, USA

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School of Civil Engineering, Guangzhou University, Guangzhou, China

Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation

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#Both

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*Corresponding

authors contributed equally to this work. author. Email address: [email protected]

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Highlights

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(1) A comparative study using aerobic sludge (AS), anaerobic sludge (AnS) and sulfate-

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reducing bacteria (SRB) sludge was conducted to examine their efficacy on

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ciprofloxacin (CIP) removal for the first time.

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(2) SRB sludge showed the highest CIP removal followed by AnS, and AS had the

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lowest CIP removal with nearly no biodegradation.

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(3) The role of extracellular polymeric substances (EPS) on CIP removal in AS, AnS

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and SRB sludge was elucidated for the first time.

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(4) The carboxyl, amine and hydroxyl functional groups were mainly involved in

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binding the CIP with EPS.

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(5) EPS in SRB sludge showed higher capacity, affinity and strength to CIP adsorption

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than those of AS and AnS.

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Abstract

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Extracellular polymeric substances (EPS) of microbial sludge play a crucial role

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in removal of organic micro-pollutants during biological wastewater treatment. In this

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study, we examined ciprofloxacin (CIP) removal in three parallel bench-scale reactors

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using aerobic sludge (AS), anaerobic sludge (AnS) and sulfate-reducing bacteria (SRB)

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sludge. The results showed that the SRB sludge had the highest specific CIP removal

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rate via adsorption and biodegradation. CIP removal by EPS accounted up to 35. 6 ±

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1.4%, 23.7 ± 0.6% and 25.5 ± 0.4% of total removal in AS, AnS and SRB sludge

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systems, respectively, at influent CIP concentration of 1,000 μg/L, which implied that

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EPS played a critical role in CIP removal. The binding mechanism of EPS on CIP

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adsorption in three sludge systems were further investigated using a series of batch tests.

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The results suggested that EPS of SRB sludge possessed stronger hydrophobicity

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(proteins/polysaccharides (PN/PS) ratio), higher availability of adsorption sites

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(binding sites (n)), and higher binding strength (binding constant (Kb)) between EPS

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and CIP compared to those of AS and AnS. The findings of this study provide an insight

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into the role of EPS in biological process for treating CIP-laden wastewaters.

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Keywords: Sulfate-reducing bacteria (SRB), Aerobic sludge (AS), Anaerobic sludge

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(AnS), Extracellular polymeric substances (EPS), Ciprofloxacin (CIP), Adsorption

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1. Introduction

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In recent years, extensive use of antibiotics for protecting humans and animals

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against infectious diseases, has caused a wide spread of antibiotics in the

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environment.1,2 Ciprofloxacin (CIP), a broad-spectrum fluoroquinolone antibiotic, is

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largely used by humans and is widely recommended by veterinarians around the world.3

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CIP use in China was reported to be about 5,340 metric tons in 2013,2 which was the

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second highest among all the fluoroquinolone antibiotics use in China. Importantly, up

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to 72% of CIP was found to exit the target subjects without being metabolized.4 The

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presence of even low levels of antibiotics in the environment has been reported

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responsible for the occurrence of antibiotic resistance genes in the environment and

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subsequent potential threat to both human beings and other species.5,6 CIP and the

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associated resistance genes have been frequently detected in municipal wastewater

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treatment plants’ effluent, various water bodies, and in soil and sediments.1,2,7,8 Thus,

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CIP, as a typical quinolone antibiotic, has attracted significant attention in recent

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years.2,6,9

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Sulfur cycle integrating carbon, nitrogen and phosphorous removal has drawn

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significant research attention in recent years.10,11 The use of sulfate-reducing bacteria

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(SRB) presents an opportunity as an energy-efficient biological process for the

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treatment of sulfur-laden industrial and municipal wastewaters.10,12 For example,

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Sulfate reduction Autotrophic denitrification and Nitrification Integrated (SANI),12

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Flue Gas Desulfurization-SANI (FGD-SANI),13 and Denitrifying Sulfur-assisted

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Enhanced Biological Phosphorous Removal (DS-EBPR)14 are some of the examples of


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SRB-mediated wastewater treatment processes. Recently, SRB sludge system has also

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been employed to treat sulfate-laden pharmaceutical wastewaters, which showed high

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tolerance of SRB against pharmaceutical compounds.15,16 Jia et al. reported that

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sulfamethoxazole could be effectively removed via adsorption and biodegradation by

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SRB-enriched sludge in an anaerobic up-flow reactor.15 Moreover, CIP was also

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effectively removed in SRB sludge system.16 Thus, SRB sludge system shows a great

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potential for antibiotics removal.

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The fate of antibiotics in wastewater treatment plants (WWTPs) has been

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extensively examined in recent years, and adsorption was found to play a critical role

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in their removal, especially in aerobic and anaerobic sludge systems.7,8,17,18 Several

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studies reported that sorption was the primary removal pathway of fluoroquinolones

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(FQs) (including CIP) in an activated sludge process.18,19 Similar findings were also

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reported in an anaerobic sludge system.20,21 In biological wastewater treatment process,

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extracellular polymeric substances (EPS), a mixture of high molecular weight polymers

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produced by microorganisms, play an important role in organic micro-pollutants

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removal.22 Proteins, as the main component in EPS, provide binding sites for the

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adsorption due to the presence of diverse functional groups, such as carboxyl, amine

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and hydroxyl groups, and hydrophobic regions.22,23 Many organic micro-pollutants

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including antibiotics (such as sulfamethazine, sulfamerazine, sulfadiazine and

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tetracycline) can be adsorbed by EPS due to the presence of above-stated functional

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groups and the hydrophobic regions.24,25,26 Although EPS play an important role in the

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removal of organic micro-pollutants, there are limited studies on the role of EPS on CIP



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removal and relevant adsorption mechanism in different biological sludge systems, e.g.,

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activated sludge (AS), anaerobic sludge (AnS) and SRB sludge.

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Therefore, the objectives of this study were to evaluate the removal of CIP in

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three parallel bench-scale reactors using AS, AnS and SRB sludge, and to examine the

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role and mechanism of EPS on CIP removal in these sludge systems. The adsorption

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mechanism of CIP by EPS were investigated using UV-visible spectral and the three-

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dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy

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technologies. The functional groups binding CIP onto EPS were identified through

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Fourier transform infrared (FTIR) spectra analysis. The findings of this study provided

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insights into the adsorption mechanism of CIP by EPS in AS, AnS and SRB sludge

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systems for the first time.

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2. Materials and methods

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2.1 Reactors setup and operation of AS, AnS and SRB sludge systems

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Three bioreactors, namely sequencing batch reactor (Figure S1(A) in

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Supporting Information (SI)), up-flow anaerobic sludge blanket (UASB) (Figure S1(B)

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in SI) and sulfate-reducing up-flow sludge bed (SRUSB) (see Figure S1(C) in SI) were

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operated side-by-side for 83 days at different stages to examine CIP removal using AS,

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AnS and SRB sludge, respectively (details of reactors operating conditions are

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presented in pages S7-S8 in SI). The reactors were fed with the same synthetic

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wastewater (see Tables S2, S3, and S4 for wastewater characteristics) without CIP for

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the first 20 days to acclimate AS, AnS and SRB sludge to synthetic wastewater followed


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by another 63 days of operation at different influent CIP concentrations (99% purity,

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Saint Louis, MO, USA): 100 ± 6.8 μg/L from Days 21 to 41; 500 ± 9.6 μg/L from Days

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42 to 62; and 1,000 ± 8.4 μg/L from Days 63 to 83. In each stage, the reactors were

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allowed to reach steady state (see page S9 in SI for details) before collecting samples

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for all the analyses. The CIP removal efficiency and specific removal rate as well as

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CIP adsorption by sludge and biodegradation at different stages of reactors operation

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were determined (see pages S9-S10 in SI for details).

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2.2 CIP adsorption by inactivated AS, AnS and SRB sludge

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A series of batch experiments were conducted to evaluate the CIP adsorption by

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AS, AnS and SRB sludge. The well-adapted AS, AnS and SRB sludge samples were

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taken on Day 83 from the sequencing batch reactor, and UASB and SRUSB reactors,

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respectively operating under steady state condition, washed three times with 10 mM

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phosphate buffer (pH=7.2), and then placed into a series of identical serum bottles for

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each sludge with average suspended solids (SS) concentration of 1.0 g/L in each bottle.

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CIP content of the sludge samples was below the detection limit. Additionally, 0.1%

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(w/v) sodium azide (NaN3) (JSK, Qingdao, China) was added into each serum bottle to

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inhibit the microbial activity for possible biodegradation of CIP by AS, AnS and SRB

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sludge.24,27 The control (without NaN3) and adsorption tests (with NaN3) showed that

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NaN3 addition did not significantly affect the EPS production during 24 hrs batch

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experiments (Figure S2 in SI). The adsorption experiments were carried out for 24 hrs

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in a series of serum bottles with total mixed liquor volume of 500 mL in each bottle and



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at different initial CIP concentrations of 100, 300, 500, 1,000, 3,000 and 5,000 μg/L.

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Different initial CIP concentrations were used to investigate the adsorption isotherms

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of CIP by AS, AnS and SRB sludge. Each serum bottle was wrapped with aluminum

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foil to avoid photodegradation. The serum bottles containing AS were initially aerated

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to achieve initial DO concentration of 4.0 mg/L, and the serum bottles were kept

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uncapped during the batch experiments. The serum bottles containing AnS and SRB

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sludge were purged with pure nitrogen gas (>99.99% purity) for 30 min before the start

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of batch test and then tightly sealed to establish anaerobic condition. The mixing was

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achieved by placing the serum bottles on the magnetic stirring plate (IKA RCT,

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Germany) at 200 rpm. In the batch experiments, initial pH was maintained at 7.0 ± 0.1

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by adding 0.1 mM HCl/NaOH solution using a pH controller (Mettler Toledo, OH, USA)

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for all three sludge samples. During the experiment, 5 mL of mixed liquor sample was

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regularly withdrawn from each serum bottle. The serum bottle experiments were

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conducted in triplicate at each CIP concentration.

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The adsorption of CIP by sludge system (EPS+Sludge) was contributed by both

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EPS and Sludge (after EPS extraction). The EPS were considered to be the tightly

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bound on the basis of the extraction method.28 In order to quantify the CIP adsorption

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by EPS and Sludge respectively, the mixed liquor samples taken from each serum bottle,

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were centrifuged at 1,793 × g for 15 min. The supernatant was used to determine the

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CIP concentration in the aqueous phase after filtering through a 0.22-μm membrane

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filter. The residual sludge pellets were used to extract the EPS using modified heating

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method28 (see pages S10-S11 in SI for details). The CIP in aqueous phase was


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quantified first to obtain the total adsorption by sludge system (EPS+Sludge). The CIP

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in the extracted EPS solution was referred to as the CIP adsorbed by EPS. The CIP

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adsorbed by Sludge was then determined by subtracting the CIP adsorbed on EPS from

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the total CIP adsorbed on the sludge system (EPS+Sludge). The removal efficiency and

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the specific adsorption (qt) of CIP were then determined (see page S11 in SI for details).

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The equilibrium adsorption capacities of CIP onto AS, AnS, and SRB sludge samples

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at different CIP concentrations (100 to 5,000 μg/L) in batch experiments were

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determined, and three adsorption isotherm models (Freundlich, Linear and Langmuir)

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were used to fit the experimental data (see pages S11-S12 in SI for details).

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2.3 CIP and EPS binding experiments

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To investigate the adsorption mechanism of CIP by EPS extracted from three

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sludge samples (i.e., AS, AnS and SRB sludge), the binding experiments were

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conducted at different CIP concentrations. The EPS were extracted from the AS, AnS

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and SRB sludge samples obtained on Day 20 from sequencing batch reactor, UASB

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and SRUSB reactors, respectively. Crude EPS was purified by polyethersulfone

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ultrafiltration membrane with 5,000 molecular weight cut off (Millipore Co., USA) to

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remove ions and small molecules.24 The retentate was lyophilized to EPS powder using

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freeze dryer (ALPHA, Marin Christ, Germany), and stored in a desiccator. Before the

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binding experiments, the EPS powder referred to as pristine EPS was weighed to

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prepare 50 mg/L of EPS solution. This EPS concentration was adopted based on the

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EEM florescence spectra that provided the best resolution as shown in Figures S3, S4



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and S5 in SI. The compositions of EPS derived from three sludge samples used in the

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binding experiments were characterized (see Table S5 in SI). Different volumes of CIP

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stock solution were added into the EPS solution to obtain different initial CIP

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concentrations (0, 50, 100, 500, 1,000, 2,000, 3,000, 4,000 and 5,000 μg/L). The initial

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pH was manually adjusted to 7.0 ± 0.1 by adding 0.1 mM of HCl/NaOH solution. The

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EPS-CIP solution in each tube was mixed for 10 min using oscillator (IKA, Vortex,

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Germany), and then placed in dark for 4 hrs at 25 ± 0.5 oC to achieve adsorption

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equilibrium before spectral analyses.24

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2.4 Analytical methods

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Chemical analyses

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The influent, effluent and sludge samples from sequencing batch reactor, UASB

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and SRUSB reactors, and the mixed liquor samples from the batch experiments were

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regularly collected for routine chemical analyses (see page S13 in SI for more details).

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CIP in aqueous phase (influent and effluent), and in EPS and sludge samples were

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determined by an ultra-performance liquid chromatography (UPLC) equipped with a

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diode array detector (Dionex UltiMate 3000, CA, USA) using an acclaim120 C18

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column (2.1×150 mm, 3 μm, Dionex, CA, USA) (details of CIP analysis are given in

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pages S13-S14 in SI). The biogas collected from the UASB reactor was analysed for its

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compositions using portable firedamp analyser (GEM 5000, Geotech, England).

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Spectral analyses 7 ACS Paragon Plus Environment

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The UV spectroscopy was employed for preliminary analysis of the interaction

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between EPS in three sludge samples and CIP in batch experiments. The UV-visible

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absorption spectra of EPS, CIP and EPS-CIP complex were measured using a UV-2700

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spectrophotometer (Shimadzu Co., Japan) with scanning wavelength ranging from 200

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to 800 nm at an increment of 1 nm. Three-dimensional excitation-emission matrix

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fluorescence quenching technology was used to further investigate the CIP adsorption

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processes by EPS extracted from AS, AnS and SRB sludge samples in batch

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experiments. The 3D-EEM fluorescence spectra of EPS before and after binding with

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different concentrations of CIP were obtained using a three-dimensional fluorescence

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spectrophotometer (Aqualog- UV-800-C, HORIBA, Japan) (details of EEM spectral

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analysis are given in pages S14-S15 in SI).

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To identify the functional groups in EPS extracted from the three sludge

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samples responsible for binding CIP in batch experiments, Fourier transform infrared

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(FTIR) spectra analysis was performed to characterize the chemical groups of EPS

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before and after binding with CIP (details of sample preparation for FTIR spectra

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analysis are given in page S15 in SI).

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Extracellular polymeric substances (EPS) characterization

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In this study, we examined the effects of CIP concentration on variations in

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EPS compositions of AS, AnS and SRB sludge. The AS, AnS and SRB sludge samples

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from sequencing batch reactor, UASB and SRUSB reactors, respectively, were

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collected on Day 0 (Seed), Day 20 (0 µg/L of CIP), Day 41 (100 µg/L of CIP), Day 62



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(500 µg/L of CIP), and Day 83 (1,000 µg/L of CIP) for EPS characterization through

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chemical and spectra analyses. The modified Lowry method, phenol-sulphuric acid

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method and anthrone method were used to measure the proteins (PN)，polysaccharides

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(PS) and humic acid (HA) contents, respectively in EPS.29,30

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Statistical analyses

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All the tests were conducted in triplicate, and the results were expressed as mean

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± standard deviation. Statistical analyses were conducted to examine the significant

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difference (statistically) in data obtained from the four stages of reactors operation.

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Multiple comparisons were based on one-way analysis of variance (ANOVA) using

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SPSS 16.0 (IBM, Armonk, NY).31 The least significant difference (LSD, p0.05) (Figure S6). In consistent with

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this study, Wang et al. also reported that FQs (ciprofloxacin, enrofloxacin, lomefloxacin,

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norfloxacin, and ofloxacin) with total concentration of 500 μg/L did not inhibit the

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activity of aerobic sludge.19 In another study with AS , Kummerer et al. also observed

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that the majority of microorganisms were not affected by FQs, despite a broad-spectrum

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of pathogenic bacteria targeted by FQs.33 However, in AnS using UASB reactor, both

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COD removal efficiency and methane yield decreased perpetually with increasing

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influent CIP concentration. For example, in Stages 2, 3 and 4, both COD removal

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efficiency and methane yield reduced by 1.7 ± 0.2% (p

Understanding the role of extracellular polymeric substances (EPS) on

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