oXiurong Si a, Xiangchun QuanaKey Laboratory of Water and Sediment Scie

Environment Simulation, School of Environme

Haidian District, Beijing 100875, China

tal Eng

th polysaccharide and

e correlated with the

amino acids and nor-

d resistant microbial

td. All rights reserved.

self-aggregate to form bioflocs or microbial clusters, all of

which could be regarded as microbial aggregates. In the field

of environment research, adverse biofilms or other microbial

s. For example, bio-

e blockage andmetal

2012); microbial ag-

gregates are inherently resistant to antimicrobial agents and

their existencewill increase the difficulty ofwater disinfection

and the amount of disinfectants used (Kollu and Ormeci,

2012). Therefore, it is necessary to develop methods to

* Corresponding author. Tel./fax: 86 10 58802374.E-mail address: xchquan@bnu.edu.cn (X. Quan).

Available online at www.sciencedirect.com

ScienceDirect

.e ls

wat e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7e2 5 31. Introduction

Microorganisms tend to attach to surfaces to form biofilms or

aggregates may result in lots of problem

films can cause membrane pollution, pip

surface corrosion (Bixler and Bhushan,Extracellular polymeric substances copy analysis revealed that norspermidine could directly interact wi

caused the disappearance of an IR band at 1152 cm1 that may b

functional group CeOeC. Overall, the combined application of D-

spermidine offers an effective approach to disassemble large an

aggregates.

2014 Elsevier LMicrobial aggregates

Disassembly

after treatment with D-Tyr and norspermidine as could be seen from the increase in surface

negative charge and decrease in cell hydrophobicity. Fourier transform infrared spectros-bDepartment of Civil and Environmen

a r t i c l e i n f o

Article history:

Received 9 October 2013

Received in revised form

27 January 2014

Accepted 2 February 2014

Available online 11 February 2014

Keywords:

D-tyrosine

Norspermidine0043-1354/$ e see front matter 2014 Elsevhttp://dx.doi.org/10.1016/j.watres.2014.02.007a,*, Qilin Li b, Yachuan Wua

nces of Ministry of Education, State Key Laboratory of Water

nt, Beijing Normal University, No. 19, Xinjiekouwai Street,

ineering, Rice University, Houston, TX 77005, USA

a b s t r a c t

The increasing threat of microbial aggregates in many fields highlights the need to develop

methods to promote their disassembly. This study investigated the coupled effects of D-

tyrosine (D-Tyr) and norspermidine on the disassembly of a type of old-aged (more than 6

months), large (about 900 mm) microbial aggregate formed by mixed culture. Results

showed that D-Tyr and norspermidine acting together effectively triggered the disassembly

of microbial aggregates, with disassembly ratio enhanced by 30e164% compared to the

control at the concentration of 50e500 mM of D-Tyr and norspermidine. D-Tyr and nor-

spermidine reduced the content of extracellular protein and polysaccharide in microbial

aggregates and altered the matrix structure of extracellular polymeric substances as

confirmed by a confocal laser scanning microscope. The microbial aggregates lost stabilityaggregates

disassembly of large, old-aged microbial

Effects of D-amino acids and n

journal homepage: wwwier Ltd. All rights reservedrspermidine on the

evier .com/locate/watres.

these purposes, such as chlorine, nano-silver and some

wat e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7e2 5 3248macromolecule antibacterial agents (Arciola et al., 2012).

However, most of these compoundsmay produce side effects.

Recent work has demonstrated that some bacteria can pro-

duce signaling molecules which could serve as biofilm disas-

sembly factors to trigger or mediate the process of biofilm-

disassembly (Kolodkin-Gal et al., 2010). D-amino acids and

norspermidine were identified from the supernatants of

disassembled biofilms and reported to be two of these

important factors (Kolodkin-Gal et al., 2012). Bacteria can

synthesize D-amino acids in stationary phase, which can

regulate the chemistry of the cell wall through

reducing the production of peptidoglycan (Lam et al., 2009).

Different D-amino acids have a different activity in inhibiting

biofilm formation, amongwhich D-tyrosinewas reported to be

more effective than other D-amino acids such as D-tryptophan

and D-leucine (Kolodkin-Gal et al., 2010). D-amino acids can

mediate biofilm disassembly by causing the release of the

protein component of the matrix in bacteria, while nor-

spermidine can interact with exopolysaccharide (Kolodkin-

Gal et al., 2012). A mixture of D-amino acids and norspermi-

dine was reported to be more effective in breaking down

existing biofilms than D-amino acids or norspermidine alone

(Kolodkin-Gal et al., 2012). Although the effects of D-amino

acids or norspermidine on disassembly of biofilms have been

well studied with short-term biofilms by pure strains, the

problem whether they can act together to trigger the disas-

sembly of long-lived, large microbial aggregates by mixed

culture remains unknown and deserves further study because

many microbial aggregates exist as complex polymicrobial

colonizations (Quinn et al., 2013).

Thus, this study aimed to understand the coupled effects of

D-tyrosine (D-Tyr), a typical D-amino acid, and norspermidine

on the disassembly of old-aged, large microbial aggregates,

which formed through self-aggregation of activated sludge in

a bioreactor and stabilized in size and shape for more than 6

months. Such microbial aggregates can be considered to be a

special case of biofilms. In this study, disassembly of the mi-

crobial aggregates under the treatment of different concen-

trations of D-Tyr and norspermidine was investigated;

possible disassembly mechanisms were explored through

investigating the changes of component, matrix structure and

functional groups of extracellular polymeric substances in

microbial aggregates. The results obtained here will promote

understanding the roles of these self-produced factors in

mediating the disassembly of undesirable microbial aggre-

gates and their potential use in microbial aggregation control.

2. Materials and methods

2.1. Microbial aggregates disassembly experimentinhibit the formation of undesirable bioaggregates and pro-

mote their disassembly. The main strategies to prevent bio-

film formation are to clean and disinfect regularly or

incorporate antimicrobial products into surface materials.

Various types of chemical compounds have been used forMicrobial aggregates, used as the targets of disassembly ex-

periments, were collected from an activated sludge reactortreating synthetic wastewater. These microbial aggregates

showed a stable granular structure with a mean diameter

(920 30 mm). The microbial composition of the aggregateswas characterized using the method of Polymerase Chain

ReactioneDenaturing Gradient Gel Electrophoresis (PCR-

DGGE) described by Ma et al. (2012). The DGGE band profile for

the microbial granule and the Blast results for sequences of

dominant genes bands on it were presented as supplementary

materials (Fig. S1 and Table S1). The microbial aggregates

mainly include Amaricoccus macauensis strain, Leifsonia sp.,

Microbacterium sp., Mesorhizobium sp., Burkholderia cepacia, Ali-

cycliphilus sp. and Acidovorax sp. Disassembly experiments

were conducted in 50 mL of vials with a reaction volume of

40 mL as follows: microbial aggregates collected for experi-

ment were first washed with phosphate buffer solution (PBS);

the vials were filled with a certain amount of substrate, mi-

crobial aggregates (final concentration of 1 g Volatile Sus-

pended Solid (VSS)/L) and spiked with D-Tyr and

norspermidine (the final concentrations of 50e500 mM); the

mixture was incubated at 180 rpm and 25 C for 48 h; largemicrobial aggregates remained in mixture were separated

from the escaped small bioflocs or planktonic cells by settling

for 2 min; the above separated two parts were collected

respectively through centrifugation and washed with a buffer

solution; the bioflocs collected were treated with alkali and

heat for complete cell lysis according to themethod described

by Rocher et al. (2001); finally, biomass of the bioflocs was

quantified in terms of Total Organic Carbon (TOC) with an

Elementar vario TOC analyzer (Elementar, Germany). All the

tests were carried out in triplicate. The components of sub-

strate were: glucose 2000 mg/L, NaHCO3 1000 mg/L, MgSO4200 mg/L, NaCl 200 mg/L, CaCl2 20 mg/L, NH4Cl 600 mg/L and

KH2PO4 88 mg/L.

After disassembly experiment, particle size distribution of

themicrobial aggregates wasmeasured by a laser particle size

analysis system with a measuring range of 0e2000 mm (Mal-

vern MasterSizer Series 2000, Malvern Instruments Ltd., Mal-

vern). The cell surface hydrophobicity of the microbial

aggregates after exposing to the mixture of D-Tyr and nor-

spermidine was determined with the method described by

Rosenberg et al. (1980). The surface zeta potential of bio-

aggregates was measured using a Zeta-potential Analyser

(ZETASIZER nano series Nano-ZS90).

2.2. EPS extraction, chemical analysis and CLSMobservation

The effects of D-Tyr and norspermidine on extracellular

polymeric substances production (EPS) in microbial aggre-

gates were determined to reveal the possible disassembly

mechanisms. Extracellular polysaccharide (PS) and protein

(PN) were extracted and quantified using themethods of Xuan

et al. (2010). Fourier transform infrared spectroscopy (FTIR)

analysis was employed to study the interactions of D-Tyr and

norspermidine with functional groups of EPS according to the

following procedure: the extracted EPS solution was added by

D-Tyr, norspermidine or their mixture and incubated for

30 min; the respective samples were freezed-dried for 48 h,and then prepared as a mixture of 1 mg sample and 100 mg

potassium bromide (KBr, IR grade); the mixed samples were

ground, homogenized and compacted to discs under high

pressure for FTIR analysis. The FTIR spectra were collected by

a nicolet iS5 FTIR spectrometer within the range

500e2000 cm1.The EPS distribution and structure in microbial aggregates

after exposing to D-Tyr and norspermidine were also observed

under a confocal laser scanning microscope (CLSM, Carl Zeiss

LSM 510, Germany) through staining with fluorescence

probes. At least ten random selected samples after exposure

experiments were observed with the CLSM. Microbial aggre-

gates after frozen at20 Cwere sectioned into 30 mmsectionswith a cryomicrotome (Leica CM3050S, Germany), and then

stained with fluorescently labeled probes fluorescein isothio-

cyanate (FITC) (Invitrogen, Carlsbad, CA, USA) and conca-

navalin A (Con A) (Invitrogen, Carlsbad, CA, USA) conjugates

respectively according to the methods of Chen et al. (2007).

The excitation/emission wavelengths for the two probes were

488/500550 nm for FITC and 543/550600 nm for Con Aconjugates. The software LSM 510 was used for image

analysis.

3. Results and discussion

Compared to the control, the relatively disassembled biomass

increased by 30 2.8% at the mixture concentration of 50 mMfor each compound, but it increased to 164.0 3.8% when theconcentration attained 500 mM.

The change of size distribution for themicrobial aggregates

after exposing to the mixture of D-Tyr and norspermidine was

also investigated (Fig. 2). Results showed that the fraction of

small size bioflocs increased apparently after treatment with

the mixture. For the control, the volume percentage of parti-

cles smaller than 200 mm accounted for 24.4%, while it

increased to 29.2% and 37.2% respectively, at the mixed con-

centration of 50 mMand 500 mM (each compound). On the other

hand, the microbial aggregates at the size of 800 mm took the

largest fraction (6.2%) for the original samples, but it was

replaced by the microbial aggregates of 670 mm after exposing

to the two compounds. All these data indicated that combined

application of D-Tyr and norspermidine can effectively pro-

mote the disassembly of the long-lived, large microbial ag-

gregates of mixed culture, which led to an increase fraction of

small size bioflocs and decrease of larger ones.

3.2. The changes of cell surface properties

wat e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7e2 5 3 249Fig. 1 e Relative biomass disassembled from the microbial

aggregates after exposure to different concentrations of D-3.1. Disassembly of microbial aggregates by D-Tyr andnorspermidine

The effects of D-Tyr and norspermidine on the disassembly of

microbial aggregates were investigated at different concen-

trations and the results were presented in Fig. 1. It showed

that the biomass disintegrated from the tested microbial ag-

gregates increased significantly after exposing to D-Tyr and

norspermidine, and the relatively disassembled biomass

increased with the increase of their concentrations.tyrosine (a) and norspermidine (b). Error bars represent

standard deviations of triplicate tests.It is well-known that cell surface properties, especially cell

surface hydrophobicity and charge, play important roles in

maintaining the stability ofmicrobial aggregates. The changes

of the two parameters were determined for the microbial ag-

gregates after disassembly experiments (Fig. 3). Compared to

the control group, the cell hydrophobicity of microbial aggre-

gates declined by 1.7e15.2% at the above tested concentra-

tions. Hydrophobic binding force is very important to hold the

aggregated bacteria tightly together and high cell hydropho-

bicity can strengthen the structure and stability of microbial

aggregates (Tay et al., 2001). The decrease of the cell hydro-

phobicity could reduce cell-to-cell interaction and promote

the disassembly of microbial aggregates (Ni et al., 2009).

Fig. 2 e The change of particle size distribution of microbial

aggregates after exposure to D-tyrosine (D-Tyr) and

norspermidine. The curves represent average of triplicate

samples and the standard deviations were obtained within0.30. Error bars represent standard deviations of triplicate

tests in the inset graph.

(Protein) and PS (Polysaccharide) in microbial aggregates after

treatment with different concentrations of D-Tyr and nor-

wat e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7e2 5 3250Furthermore, in a thermodynamic sense, the decrease of cell

surface hydrophobicity could lead to an increase in the excess

Gibbs energy of the surface, which will in turn inhibit the self-

aggregation of bacteria from liquid phase to form a new solid

phase (Yang et al., 2004).

Fig. 3 e The changes of hydrophobicity and surface charge

of microbial aggregates after exposure to D-tyrosine (a) and

norspermidine (b). Error bars represent standard

deviations of triplicate tests.Surface charge is another important factor influencing

microbial aggregation and stability (Zhang et al., 2007). It could

be seen from Fig. 3 that the zeta potential increased with the

increase of D-Tyr and norspermidine concentrations. The

control showed a zeta potential of 25.6 0.3 mV, while thesamples treated with 500 mM (each) of D-Tyr and norspermi-

dine had a zeta potential of35.6 0.2mV. According to DLVOtheory, increased negative chargewould lead to the increasing

dispersion due to the increase of electrical repulsion between

cells, which would repress cell-to-cell approach and interac-

tion (Mikkelsen and Keiding, 2002). Similar results were re-

ported by Li et al. (2006), who found that the surface charge in

terms of zeta potential tended to decrease with sludge ag-

gregation and granular formation. Hence, the extent of

disassembly would be further strengthened (Fig. 1) as a result

of the increased negative charge. The decrease of hydropho-

bicity and increase of surface negative charge also indicated

that the microbial aggregates were in a great unstable status

after treatment with D-Tyr and norspermidine, which might

further trigger or promote disintegration of microbial

aggregates.

3.3. Effects of D-Tyr and norspermidine on EPSproduction in microbial aggregates

EPS is the main components of microbial aggregates and can

contribute to the formation of microbial aggregates and

maintenance their stability and integrity (Flemming andWingender, 2001). Fig. 4 showed the relative amount of PN

Fig. 4 e Effects of D-tyrosine (a) and norspermidine (b) on

the relative production of extracellular protein (PN) and

polysaccharide (PS) in microbial aggregates. Error bars

represent standard deviations of triplicate tests.spermidine. Results showed that D-Tyr and norspermidine

caused a significant reduction of PN and PS at the tested

concentrations, and this reduction was more significant at

relatively high concentrations. As compared to the control, PS

in the microbial aggregates decreased by 25.8 1.1% and PNdecreased by 40.8 0.6% after exposing to 500 mM of themixture.

The spatial distributions of PN and PS in microbial aggre-

gates were observed with a confocal laser scanning micro-

scopy (CLSM) (Fig. 5). For the control microbial aggregates,

exopolysaccharide and protein distributed in whole sections

of granular microbial aggregates, and formed an interwoven

meshwork in the matrix, which helps hold cell together (Fig. 5

a1-a4). After treatment with D-Tyr and norspermidine, PN and

PS density declined significantly especially in the outer layers,

and their inter connections became loose and the network

structure collapsed, which promoted cell to escape from the

aggregates and return to planktonic status (Fig. 5 b1eb4, c1ec4

and d1ed4). Overall, the mixture of D-Tyr and norspermidine

not only reduced EPS content but also altered PN and PS ma-

trix structure in the microbial aggregates.

3.4. Interactions of D-Tyr and norspermidine with EPS

The above study indicated that the combination of D-Tyr and

norspermidine resulted in the change of EPSmatrix. Thus, the

effects of the two substances on the chemical functional

groups of EPS were further investigated using FTIR (Fig. 6).

Fig. 5 e EPS distributions in microbial aggregates before (a1-a4)

of D-tyrosine and norspermidine each at a concentration of 500

(polysaccharide, blue, a1, b1, c1 and d1), and FITC (protein, green

b3, c3 and d3. Overlay of polysaccharide, protein, and phase co

interpretation of the references to color in this figure legend, th

Fig. 6 e FTIR image showing interactions of D-tyrosine (D-

Tyr) and norspermidine with functional groups of

extracellular polymeric substances.

wat e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7e2 5 3 251FTIR spectra of EPS exhibited a few characteristic bands rep-

resenting several functional groups, such as amino, carbonyl

and carboxyl, which were similar to results of FTIR spectra for

EPS extracted from microbial granules (Mu et al., 2012). After

norspermidine treatment, the band at 1152 cm1 related to thestretching vibration of CeOeC from polysaccharide dis-

appeared (Xu and Liu, 2008). This indicated that poly-

saccharide had a direct interaction with norspermidine and

CeOeC was the active binding site, which is consistent with

the finding obtained from a pure culture experiment

(Kolodkin-Gal et al., 2012). Polysaccharides often contain

neutral sugars with polar groups or negatively charged resi-

dues in the secondary structure (Sutherland, 2001). It was re-

ported that amines in norspermidine could interact directly

and specifically with such charged or polar groups (Kolodkin-

Gal et al., 2012). Recently, a library of compounds that struc-

turally mimicked norspermidine was synthesized chemically,

which inhibited biofilm formation and disrupted existing

biofilms by Bacillus subtilis and Staphylococcus aureus through

binding to negatively charged or possibly polar groups

and after (b1-b4, c1-c4 and d1-d4) treatment with a mixture

mM. The sections were simultaneously stained with Con A

, a2, b2, c2 and d2). Phase contrast images are shown in a3,

ntrast images are shown in a4, b4, c4 and d4. (For

e reader is referred to the web version of this article.)

through coulombic attraction and hydrogen bonding (Bottcher

et al., 2013). This result also explained the PS reduction after

treatment with the mixture. The new band at 1434 cm1 cor-responded to the vibration of NeH from norspermidine.

However, treatment of the EPS with D-Tyr had little effect on

the functional groups of EPS, indicating that there was no

direct chemical reaction between them. Therefore, it can be

inferred that D-Tyr and norspermidine might act by different

mechanisms to cause the decrease of EPS in microbial aggre-

gates. D-Tyr can adjust extracellular protein through blocking

adhesion proteins from localizing at cell wall (Hochbaum

et al., 2011). The mechanism could partly explain the

disassembly throughmultiplemechanismsmentioned above.

Different to D-Tyr, amine in norspermidine was believed to

wat e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7e2 5 3252decrease of EPS in microbial aggregates caused by D-Tyr.

Several new bands at 1362 cm1, 1326 cm1, 1266 cm1,1043 cm1 and 876 cm1 also appeared after D-Tyr treatment.They were characteristic bands of the dCeO, dCeH stretches,

deformation vibration of C]O, nCeOH and aromatic ring of

the D-Tyr, respectively (Badireddy et al., 2010). The band at

1434 cm1 in the spectrum of the sample treated with D-Tyrcould be assigned to the nC-C vibration of the aromatic ring of

D-Tyr (Hellwig et al., 2002; Ramaekers et al., 2005).

3.5. Possible mechanisms of microbial aggregatesdisassembly by D-Tyr and norspermidine

EPS is responsible for the cohesive forces in microbial aggre-

gation. Cells in the microbial aggregates are held together by

EPS matrix (Flemming and Wingender, 2001). Microbes can

hardly aggregate when the metabolic EPS synthesis is blocked

(Adav et al., 2008). In this study, a significant reduction of EPS

and an evident change of EPS matrix structure in microbial

aggregates were observed, which might be the important

reason for the disassembly of microbial aggregates, as shown

in Fig. 7. In addition, the decrease of hydrophobicity and in-

crease of surface negative charge might further deteriorate

the stability of microbial aggregates and trigger or promote

their disintegration. As D-Tyr and norspermidine did not

inhibit cell growth (data not shown) even at millimolar con-

centrations, so this factor can be ruled out for the disassembly

of microbial aggregates.

Several researchers studied the possible mechanisms of D-

Tyr inhibition to cell attachment and biofilm formation. D-Tyr

was reported to have a function of modulating synthesis of

peptidoglycan in cell wall of some bacteria through incorpo-

ration into it or regulating relative enzymes activity (Lam

Fig. 7 e Schematic illustrating mechanisms of microbialaggregates disassembly caused by D-tyrosine and

norspermidine.interact with negatively charged residues or natural sugars

with polar groups in exopolysaccharides (Kolodkin-Gal et al.,

2012). In this study, norspermidine was found to interact

with the polysaccharide directly through binding to the

functional group CeOeC, whichmight be an important reason

for PS reduction. A mixture of D-Tyr and norspermidine was

more effective in reducing EPS content by acting in a com-

plementary manner.

4. Conclusions

This study for the first time demonstrated the capability of

using the mixture of D-amino acids and norspermidine as an

effective approach to trigger the disassembly of old-aged,

large microbial aggregates formed by mixed culture. The

increase of surface negative charge, decrease of cell hydro-

phobicity and remarkable reduction of EPS in microbial ag-

gregates could contribute to the disassembly of microbial

aggregates. Norspermidine could interact directly with the

PS through binding to the functional group CeOeC. D-Tyr is

supposed to reduce PN production through incorporation

into the peptidoglycan and changing protein in cell wall.

Combined application of D-amino acids and norspermidine

offers a potential approach to clean up thick, old-aged and

resistant biofilms formed on pipeline or membrane, or to

break up large and complex microbial clusters in disinfec-

tion systems.

Acknowledgmentset al., 2009). For example, Kolodkin-Gal et al. (2010) observed

D-Tyr could cause the release and disassembly of the protein

component of the matrix in a Gram-positive bacterium B.

subtilis by incorporating into the peptidoglycan and combining

with the receptor protein TapA in cell wall. D-Tyr also pre-

vented biofilm formation by another Gram-positive bacterium

S. aureus through inhibiting the accumulation of the protein

component of the matrix, although this strain has no

apparent ortholog of protein TapA (Hochbaum et al., 2011),

suggesting D-Tyr might inhibit biofilm formation through

blocking different adhesion proteins from localizing at cell

wall. Yu et al. (2012) recently found that D-Tyr was also

effective in controlling cell attachment and biofilm formation

by a model Gram-negative bacterium Pseudomonas aeruginosa

on a polyamide nanofiltration, possibly due to the changes of

peptidoglycan or other components in outer membrane or

lipopolysacchrides. In addition, D-Tyr was also reported to

inhibit the production of quorum sensing signalmolecule AI-2

in mixed culture, which might also contribute to biofilm in-

hibition (Xu and Liu, 2011). The microbial aggregates studied

here involved multiple microbial species including both

Gram-positive and Gram-negative bacteria. Therefore, D-Tyr

may work on the microbial aggregates and promote theirThis research was supported by National Natural Science

Foundation of China (No. 51178049).

Appendix A. Supplementary data

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Effects of d-amino acids and norspermidine on the disassembly of large, old-aged microbial aggregates1 Introduction2 Materials and methods2.1 Microbial aggregates disassembly experiment2.2 EPS extraction, chemical analysis and CLSM observation

3 Results and discussion3.1 Disassembly of microbial aggregates by d-Tyr and norspermidine3.2 The changes of cell surface properties3.3 Effects of d-Tyr and norspermidine on EPS production in microbial aggregates3.4 Interactions of d-Tyr and norspermidine with EPS3.5 Possible mechanisms of microbial aggregates disassembly by d-Tyr and norspermidine

4 ConclusionsAcknowledgmentsAppendix A Supplementary dataReferences