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Effects of D-amino acids and norspermidine on the

disassembly of large, old-aged microbial
aggregates
Xiurong Si a, Xiangchun Quan a,*, Qilin Li b, Yachuan Wu a
a

Key Laboratory of Water and Sediment Sciences of Ministry of Education, State Key Laboratory of Water
Environment Simulation, School of Environment, Beijing Normal University, No. 19, Xinjiekouwai Street,
Haidian District, Beijing 100875, China
b
Department of Civil and Environmental Engineering, Rice University, Houston, TX 77005, USA

article info

abstract

Article history:

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

Received 9 October 2013

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

Received in revised form

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

27 January 2014

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

Accepted 2 February 2014

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

Available online 11 February 2014

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-

Keywords:

spermidine reduced the content of extracellular protein and polysaccharide in microbial

D-tyrosine

aggregates and altered the matrix structure of extracellular polymeric substances as

Norspermidine

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

Microbial aggregates

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

Disassembly

negative charge and decrease in cell hydrophobicity. Fourier transform infrared spectros-

Extracellular polymeric substances

copy analysis revealed that norspermidine could directly interact with polysaccharide and
caused the disappearance of an IR band at 1152 cm
1 that may be correlated with the
functional group CeOeC. Overall, the combined application of

D-amino

acids and nor-

spermidine offers an effective approach to disassemble large and resistant microbial

aggregates.
2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Microorganisms tend to attach to surfaces to form biofilms or

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

aggregates may result in lots of problems. For example, biofilms can cause membrane pollution, pipe blockage and metal
surface corrosion (Bixler and Bhushan, 2012); microbial aggregates are inherently resistant to antimicrobial agents and
their existence will increase the difficulty of water disinfection
rmeci,
and the amount of disinfectants used (Kollu and O
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).
0043-1354/$ e see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.watres.2014.02.007

248

w a t e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7 e2 5 3

inhibit the formation of undesirable bioaggregates and promote their disassembly. The main strategies to prevent biofilm formation are to clean and disinfect regularly or
incorporate antimicrobial products into surface materials.
Various types of chemical compounds have been used for
these purposes, such as chlorine, nano-silver and some
macromolecule antibacterial agents (Arciola et al., 2012).
However, most of these compounds may produce side effects.
Recent work has demonstrated that some bacteria can produce signaling molecules which could serve as biofilm disassembly factors to trigger or mediate the process of biofilmdisassembly (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, among which D-tyrosine was 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 norspermidine can interact with exopolysaccharide (KolodkinGal et al., 2012). A mixture of D-amino acids and norspermidine 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 disassembly 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 microbial aggregates under the treatment of different concentrations 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 aggregates and their potential use in microbial aggregation control.

2.

Materials and methods

2.1.

Microbial aggregates disassembly experiment

Microbial aggregates, used as the targets of disassembly experiments, were collected from an activated sludge reactor

treating synthetic wastewater. These microbial aggregates

showed a stable granular structure with a mean diameter
(920  30 mm). The microbial composition of the aggregates
was characterized using the method of Polymerase Chain
ReactioneDenaturing Gradient Gel Electrophoresis (PCRDGGE) 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, Alicycliphilus 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 experiment were first washed with phosphate buffer solution (PBS);
the vials were filled with a certain amount of substrate, microbial aggregates (final concentration of 1 g Volatile Suspended 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; large
microbial 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 the method 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 substrate were: glucose 2000 mg/L, NaHCO3 1000 mg/L, MgSO4
200 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
the microbial aggregates was measured by a laser particle size
analysis system with a measuring range of 0e2000 mm (Malvern MasterSizer Series 2000, Malvern Instruments Ltd., Malvern). The cell surface hydrophobicity of the microbial
aggregates after exposing to the mixture of D-Tyr and norspermidine was determined with the method described by
Rosenberg et al. (1980). The surface zeta potential of bioaggregates was measured using a Zeta-potential Analyser
(ZETASIZER nano series Nano-ZS90).

2.2.
EPS extraction, chemical analysis and CLSM
observation
The effects of D-Tyr and norspermidine on extracellular
polymeric substances production (EPS) in microbial aggregates were determined to reveal the possible disassembly
mechanisms. Extracellular polysaccharide (PS) and protein
(PN) were extracted and quantified using the methods 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

w a t e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7 e2 5 3

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 cm
1.
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 aggregates after frozen at
20  C were sectioned into 30 mm sections
with a cryomicrotome (Leica CM3050S, Germany), and then
stained with fluorescently labeled probes fluorescein isothiocyanate (FITC) (Invitrogen, Carlsbad, CA, USA) and concanavalin 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
488/500
550 nm for FITC and 543/550
600 nm for Con A
conjugates. The software LSM 510 was used for image
analysis.

Compared to the control, the relatively disassembled biomass

increased by 30  2.8% at the mixture concentration of 50 mM
for each compound, but it increased to 164.0  3.8% when the
concentration attained 500 mM.
The change of size distribution for the microbial 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 particles smaller than 200 mm accounted for 24.4%, while it
increased to 29.2% and 37.2% respectively, at the mixed concentration of 50 mM and 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 promote the disassembly of the long-lived, large microbial aggregates of mixed culture, which led to an increase fraction of
small size bioflocs and decrease of larger ones.

3.2.

3.

249

The changes of cell surface properties

Results and discussion

3.1.
Disassembly of microbial aggregates by D-Tyr and
norspermidine
The effects of D-Tyr and norspermidine on the disassembly of
microbial aggregates were investigated at different concentrations and the results were presented in Fig. 1. It showed
that the biomass disintegrated from the tested microbial aggregates increased significantly after exposing to D-Tyr and
norspermidine, and the relatively disassembled biomass
increased with the increase of their concentrations.

Fig. 1 e Relative biomass disassembled from the microbial

aggregates after exposure to different concentrations of Dtyrosine (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 of microbial aggregates. The changes
of the two parameters were determined for the microbial aggregates after disassembly experiments (Fig. 3). Compared to
the control group, the cell hydrophobicity of microbial aggregates declined by 1.7e15.2% at the above tested concentrations. Hydrophobic binding force is very important to hold the
aggregated bacteria tightly together and high cell hydrophobicity can strengthen the structure and stability of microbial
aggregates (Tay et al., 2001). The decrease of the cell hydrophobicity 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 within
0.30. Error bars represent standard deviations of triplicate
tests in the inset graph.

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w a t e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7 e2 5 3

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.

Furthermore, 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 selfaggregation of bacteria from liquid phase to form a new solid
phase (Yang et al., 2004).
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 the
samples treated with 500 mM (each) of D-Tyr and norspermidine had a zeta potential of
35.6  0.2 mV. According to DLVO
theory, increased negative charge would lead to the increasing
dispersion due to the increase of electrical repulsion between
cells, which would repress cell-to-cell approach and interaction (Mikkelsen and Keiding, 2002). Similar results were reported by Li et al. (2006), who found that the surface charge in
terms of zeta potential tended to decrease with sludge aggregation 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 hydrophobicity 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 EPS
production 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 and

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.

Wingender, 2001). Fig. 4 showed the relative amount of PN

(Protein) and PS (Polysaccharide) in microbial aggregates after
treatment with different concentrations of D-Tyr and norspermidine. 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 PN
decreased by 40.8  0.6% after exposing to 500 mM of the
mixture.
The spatial distributions of PN and PS in microbial aggregates were observed with a confocal laser scanning microscopy (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 matrix 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 EPS matrix. Thus, the
effects of the two substances on the chemical functional
groups of EPS were further investigated using FTIR (Fig. 6).

w a t e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7 e2 5 3

251

Fig. 5 e EPS distributions in microbial aggregates before (a1-a4) and after (b1-b4, c1-c4 and d1-d4) treatment with a mixture
of D-tyrosine and norspermidine each at a concentration of 500 mM. The sections were simultaneously stained with Con A
(polysaccharide, blue, a1, b1, c1 and d1), and FITC (protein, green, a2, b2, c2 and d2). Phase contrast images are shown in a3,
b3, c3 and d3. Overlay of polysaccharide, protein, and phase contrast images are shown in a4, b4, c4 and d4. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 e FTIR image showing interactions of D-tyrosine (DTyr) and norspermidine with functional groups of
extracellular polymeric substances.

FTIR spectra of EPS exhibited a few characteristic bands representing 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 cm
1 related to the
stretching vibration of CeOeC from polysaccharide disappeared (Xu and Liu, 2008). This indicated that polysaccharide 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 residues in the secondary structure (Sutherland, 2001). It was reported that amines in norspermidine could interact directly
and specifically with such charged or polar groups (KolodkinGal et al., 2012). Recently, a library of compounds that structurally 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

252

w a t e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7 e2 5 3

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 cm
1 corresponded 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 aggregates. 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
decrease of EPS in microbial aggregates caused by D-Tyr.
Several new bands at 1362 cm
1, 1326 cm
1, 1266 cm
1,
1043 cm
1 and 876 cm
1 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 cm
1 in the spectrum of the sample treated with D-Tyr
could 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 aggregates
disassembly by D-Tyr and norspermidine
EPS is responsible for the cohesive forces in microbial aggregation. 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 increase 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 concentrations, so this factor can be ruled out for the disassembly
of microbial aggregates.
Several researchers studied the possible mechanisms of DTyr 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 incorporation into it or regulating relative enzymes activity (Lam

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

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 prevented 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 signal molecule AI-2
in mixed culture, which might also contribute to biofilm inhibition (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 their
disassembly through multiple mechanisms mentioned above.
Different to D-Tyr, amine in norspermidine was believed to
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, which might 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 complementary manner.
D-Tyr

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 hydrophobicity and remarkable reduction of EPS in microbial aggregates 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 disinfection systems.

Acknowledgments
Fig. 7 e Schematic illustrating mechanisms of microbial
aggregates disassembly caused by D-tyrosine and
norspermidine.

This research was supported by National Natural Science

Foundation of China (No. 51178049).

w a t e r r e s e a r c h 5 4 ( 2 0 1 4 ) 2 4 7 e2 5 3

Appendix A. Supplementary data

Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.02.007

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