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Characterization of extracellular polymeric substances of
aerobic and anaerobic sludge using three-dimensional
excitation and emission matrix uorescence spectroscopy
Guo-Ping Sheng, Han-Qing Yu

Laboratory of Environmental Engineering, School of Chemistry, The University of Science & Technology of China, Hefei, Anhui 230026, China
a r t i c l e i n f o
Article history:
Received 12 October 2005
Received in revised form
6 December 2005
Accepted 10 January 2006
Keywords:
Excitationemission matrix (EEM)
uorescence spectroscopy
Extracellular polymeric substances
(EPS)
Humic substances
Proteins
Sludge
A B S T R A C T
In this study three-dimensional excitationemission matrix (EEM) uorescence spectro-
scopy was applied to characterize the extracellular polymeric substances (EPS) extracted
from aerobic and anaerobic sludge in wastewater treatment. Three uorescence peaks
were identied in EEM uorescence spectra of the EPS samples. Two peaks were attributed
to the protein-like uorophores, and the third to the humic-like uorophores. The effects of
both pH and EPS concentration were signicant on EEM uorescence spectra of EPS, but the
ionic strength had no substantial effect on EEM spectra of the EPS. The differences in the
EPS uorescence parameters, e.g., peak locations, intensities and ratios of various peak
intensities, indicate the difference in the chemical structures of the EPS from various
origins. EEM spectroscopy was proven to be an appropriate and effective method to
characterize the EPS from various origins in wastewater treatment systems.
& 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In biological wastewater treatment, extracellular polymeric
substances (EPS) are produced by the microorganisms in
aerobic and anaerobic sludge when organic materials present
in wastewater are consumed. EPS are a complex high-
molecular-weight mixture of polymers (M
w
410,000) excreted
by microorganisms, produced from cell lysis and hydrolysis,
and adsorbed organic matter from wastewater. EPS are
involved in the formation of microbial aggregates, adhesion
to surfaces and occulation (Wingender et al., 1999). Further-
more, EPS are a major component of microbial aggregates for
keeping them together in a three-dimensional matrix due to
bridging with multivalent cations (Frolund et al., 1996).
Because of their crucial roles in the structure and functions
of microbial aggregates like sludge, many attempts have
been made to explore their chemical compositions (Sheng
et al., 2005) and physicochemical properties (Wilen et al.,
2003).
With the application of numerous innovative analytical
instruments, e.g., confocal laser scanning microscopy (Staudt
et al., 2004) and Fourier transform infrared spectroscopy
(Sheng et al., 2005), a large amount of information about the
structural and functional properties of EPS and their environ-
mental behavior has been obtained. EPS contain large
quantities of aromatic structures and unsaturated fatty
chains with various types of functional groups (Wingender
et al., 1999), which have uorescence characteristics. Their
intrinsic uorescence characteristics can provide information
concerning the structure, functional groups, conguration,
and heterogeneity of the components in sludge EPS. Fluores-
cence spectroscopy is very sensitive and is substantially
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0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2006.01.023

Corresponding author. Fax: +86 551 3601592.

E-mail address: hqyu@ustc.edu.cn (H.-Q. Yu).
WAT E R R E S E AR C H 40 ( 2006) 1233 1239
affected by many factors, such as solvent, pH, ionic strength,
temperature and metal ions, etc. (Reynolds and Ahmad 1995;
Mobed et al., 1996; Peuravuori et al., 2002). Fluorescence
spectroscopy of EPS under various conditions could provide
important information on their chemical compositions,
because the uorescence characteristics are functions of
structure and functional groups in molecules.
Three-dimensional excitationemission matrix (EEM) uor-
escence spectroscopy is a rapid, selective and sensitive
technique. The outstanding advantage of EEM uorescence
spectroscopy is that information regarding the uorescence
characteristics can be entirely acquired by changing excita-
tion wavelength and emission wavelength simultaneously.
Thus, because of its high sensitivity, good selectivity, and
non-destruction of samples, EEM uorescence spectroscopy
could be useful for studying the chemical and physical
properties of EPS. It can be used to distinguish the uores-
cence compounds present in the complex EPS mixtures from
various origins. EEM uorescence spectroscopy has been
successfully used to evaluate the characteristics of natural
dissolved organic matter and humic substances from various
origins (Coble, 1996; Baker 2001; Lu and Jaffe, 2001; Reynolds,
2002; Chen et al., 2003). It has been proven to be a useful
technique to differentiate the changes and transformations of
organic matter in natural environments. The main compo-
nents of the EPS samples are proteins, humic substances, and
carbohydrates (Wingender et al., 1999). Compared with
proteins and humic substances, the intensity of EEM uores-
cence spectrum of carbohydrates could be neglected (Her et
al., 2003). Thus, the uorescence signals of the EPS ere mainly
attributed to proteins and humic substances. Esparza-Soto
and Westerhoff (2001) had investigated the soluble and
readily extractable EPS fractions of activated sludge using
uorescence spectroscopy. However, no effort has been made
to compare the EPS from various origins in wastewater
treatment using three-dimensional EEM uorescence spec-
troscopy.
This work aimed at characterizing the uorescence signals
of the EPS extracted from both aerobic (dened as AE-EPS)
and anaerobic sludge (dened as AN-EPS) using EEM uores-
cence spectroscopy. The relationships between EEM spectral
characteristics and pH, EPS concentration and ionic strength
were also evaluated. The information would be valuable for
understanding the characteristics of sludge EPS from various
origins.
2. Materials and methods
2.1. Sludge
The aerobic sludge was collected from an aeration tank in the
Wangxiaoying Municipal Wastewater Treatment Plant, Hefei,
China. The anaerobic methanogenic sludge was sampled
from a pilot-scale upow anaerobic sludge blanket reactor
treating soybean-processing wastewater. The ratios of the
volatile suspended solids (VSS) to suspended solids (SS) of the
aerobic and anaerobic sludge were 66.3% and 61.5%, respec-
tively.
2.2. EPS extraction and chemical analyses
The EPS of the aerobic and anaerobic sludge were extracted
using the cation exchange resin (CER) technique (Dowex
Marathon C, 2050 mesh, sodium form, Fluka 91973) accord-
ing to Frolund et al. (1996). Both sludge samples were
harvested by centrifugation at 3000rpm for 15min respec-
tively, and then the pellets were washed twice with 100mM
NaCl solution. After that the sludge pellets were re-suspended
to a pre-determined volume and the solution was transferred
to an extraction beaker, followed by the CER addition
with a dosage of 60 g/g SS. These suspensions were
then stirred for 12h at 200rpm and 4 1C. Afterwards the
CER/sludge suspensions were settled for 3 min in order to
remove CER, and the EPS were harvested by centrifugation at
12,000rpm and 4 1C for 30min to remove remaining sludge
components. The supernatants were then ltrated through
0.45-mm acetate cellulose membranes and were used as the
EPS fraction for chemical and EEM uorescence spectral
analyses.
All other chemicals, except CER, were purchased from
Chemical Reagent Corp., Shanghai, and were of analytical
grade. The total EPS content was measured as total organic
carbon (TOC-V
CPN
analyzer, Shimadzu, Japan). The SS and
VSS of the sludge samples were determined according to the
Standard Methods (APHA, 1995).
2.3. EEM uorescence spectroscopy
EEM spectra of sludge EPS were recorded at various pH values,
ionic strengths and EPS concentrations. The experimental
conditions are listed in Table 1. The pH was adjusted
with 0.1M NaOH or HCl solutions, while the ionic strength
was adjusted with 0.1M NaCl solution and double distilled
water.
All EEM spectra were measured using a luminescence
spectrometry (LS-55, Perkin-Elmer Co., USA). EEM spectra
are a collection of a series of emission spectra over a range of
excitation wavelengths, and they can be used to identify
uorescent compounds present in complex mixtures. In
this study, EEM spectra were collected with subsequent
scanning emission spectra from 300 to 550nm at 0.5nm
increments by varying the excitation wavelength from
200 to 400nm at 10nm increments. The other two
emission spectra by the excitation wavelength at 225
and 285nm were also collected. Excitation and emission
slits were maintained at 10 nm and the scanning speed was
set at 1200nm/min for all the measurements. A 290nm
emission cutoff lter was used in scanning to eliminate
second order Raleigh light scattering. The spectrum of double
distilled water was recorded as the blank. The software
MatLab 6.5 (MathWorks Inc., USA) was employed for handling
EEM data.
EEM spectra are illustrated as the elliptical shape of
contours. The X-axis represents the emission spectra from
300 to 550nm, whereas the Y-axis is the excitation wave-
length from 200 to 400nm. Forty contour lines, as the third
dimension, are shown for each EEM spectra to represent the
uorescence intensity at an interval of 10.
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WAT E R R E S E A R C H 40 ( 2006) 1233 1239 1234
3. Results and discussion
3.1. EEM spectra of AE-EPS and AN-EPS
In this study three-dimensional EEM spectroscopy was
applied for characterizing the EPS extracted from both aerobic
and anaerobic sludge. Each EEM gave spectral information
about the chemical compositions of EPS samples. Three peaks
were readily identied from EEM uorescence spectra of the
AE-EPS and AN-EPS (Fig. 1). The rst main peak was identied
at excitation/emission wavelengths (Ex/Em) of 225/
340350nm (Peak A), while the second main peak was
identied at Ex/Em of 280285/340350nm (Peak B). The two
peaks have been described as protein-like peaks, in which the
uorescence is associated with the aromatic amino acid
tryptophan (Baker, 2001; Chen et al., 2003; Yamashita and
Tanoue, 2003; Baker and Inverarity, 2004). Compared with the
uorescence peak location of proteins reported previously
(276281/340370nm) (Baker, 2001), the locations of Peak B for
the two EPS showed a blue shift. A third peak was located
around Ex/Em 330340/420430nm (Peak C). A similar
uorescence signal has also been observed for natural
dissolved organic matter and is described as visible humic
acid-like uorescence (Coble, 1996). Compared with the
uorescence maxima of humic acids and fulvic acids (325/
452, 320/443nm) reported by Mobed et al. (1996), the locations
of Peak C for both EPS samples had a blue shift.
EEM spectra can also be employed for quantitative analysis.
Fluorescence parameters, such as peak location, maximum
uorescence intensity and different peak intensity ratios,
were extracted from EEM uorescence EEM spectra, and are
listed in Tables 24. Peak locations of the two EPS uorescence
spectra were slightly different. The locations of these peaks
for the AE-EPS were red shifted to longer wavelengths than
those of the AN-EPS. At the same pH, the ratio of Peak A/Peak
B of the AN-EPS was less than that of the AE-EPS. Such
differences imply that the uorophores presented in the two
EPS samples were from the two different sludge origins.
According to Esparza-Soto and Westerhoff (2001), three
uorescence peaks of EPS of activated sludge were found at
205215/335, 270/335 and 220275/340350nm. However, the
locations of the three uorescence peaks of AE-EPS and AN-
EPS of in the present study all showed a red shift. Such a
difference suggests that the components in the EPS extracted
in the present study were chemically different from those
investigated by Esparza-Soto and Westerhoff (2001).
3.2. EEM spectra of EPS at various pH values
EEM spectra of the AE-EPS and AN-EPS at pH 3.011.0 are,
respectively, illustrated in Figs. 2 and 3, whereas their spectral
characteristics are listed in Table 2. The environmental pH
had a substantial effect on the uorescence characteristics of
EEM spectra of the sludge EPS. Slight shifts were observed for
the peak locations of the samples at various pH values. For
the AE-EPS, the location of Peaks A, B and C at pH 11.0 was red
shifted by 9, 7 and 3 nm, respectively, compared with those at
pH 7.0. For the AN-EPS, the location of Peaks A, B and C at pH
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Table 1 Experimental conditions for EEM uorescence spectroscopic measurement
Factors pH EPS concentration (mg C/l) Ionic strength (mM)
pH AE-EPS 311 11.3 10
AN-EPS 311 7.4 10
EPS concentration AE-EPS 7 2.845.2 40
AN-EPS 7 1.829.6 40
Ionic strength AE-EPS 7 11.3 0100
AN-EPS 7 7.4 0100
Fig. 1 EEM uorescence spectra of: (a) AE-EPS; and (b) AN-EPS.
WATER RESEARCH 40 ( 2006) 1233 1239 1235
11.0 was red shifted by 5, 6, 11nm, respectively, than those at
pH 7.0. Such a shift was also observed by others (Mobed et al.,
1996), and it showed the changes in the conformations of the
components in EPS at different pH values.
The intensities of the three peaks increased with increasing
pH in a range of 3.010.0. As pH was increased to 11.0, the
intensities of Peaks A and B both decreased (Figs. 4a and b).
This result was in accordance with that of previous studies
(Pullin and Cabaniss, 1995; Reynolds and Ahmad, 1995; Mobed
et al, 1996; Patel-Sorrentino et al., 2002), in which the
uorescence intensities of humic substances or dissolved
organic matter were found to increase with increasing pH.
This might be attributed to the changes in uorescence
characteristics of the acidic functional groups in the organic
macromolecular structure, as well as the changes of their
own molecular congurations with pH (Mobed et al., 1996;
Patel-Sorrentino et al., 2002). The intensity ratios of Peak A/
Peak B for the AE-EPS and AN-EPS remained around 2.6 and
2.4, respectively, regardless of pH value. Equal peak ratios at
different pH values are likely to suggest that the uorophores
of Peaks A and B in the EPS samples were from the same
origin (Coble, 1996). However, the different values of peak
intensity ratio of the two EPS may imply that the structural
differences in the compounds are responsible for the
uorescence characteristics of the AE-EPS and AN-EPS.
The uorescence parameters of Peak C were signicantly
affected by pH, and it was also true for the cases of peak
intensity ratios A/C and B/C. For the AE-EPS, these ratios
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Table 2 Fluorescence spectral parameters of the sludge EPS samples at various pH values
pH Peak A Peak B Peak C A/B A/C B/C
Ex/Em Int.
a
Ex/Em Int.
a
Ex/Em Int.
a
AE-EPS 3 225/342 265.2 280/347 99.8 330/420.5 26.8 2.66 9.90 3.72
4 225/341 338.4 280/345 130.2 340/421.5 26.8 2.60 12.62 4.85
5 225/342 368.1 280/342.5 144.7 340/424 24.5 2.54 15.06 5.92
6 225/341.5 400.4 285/345 153.0 340/425.5 23.3 2.62 17.16 6.56
7 225/340.5 406.4 280/343 156.6 340/428.5 23.3 2.59 17.41 6.71
8 225/341.5 394.4 280/343 154.3 350/431 24.3 2.56 16.21 6.33
9 225/342 411.5 280/343 163.6 350/431.5 25.1 2.52 16.40 6.52
10 225/341.5 411.4 280/344.5 157.0 340/426 28.0 2.62 14.72 5.62
11 225/349.5 384.1 280/349.5 143.5 340/425.5 34.6 2.68 11.10 4.15
AN-EPS 3 225/346 148.8 280/352 61.9 330/427.5 14.0 2.40 10.67 4.44
4 225/346.5 182.2 280/352 74.0 330/425 14.2 2.46 12.86 5.22
5 225/345.5 235.3 280/350.5 95.2 340/425.5 14.8 2.47 15.92 6.44
6 225/345.5 261.8 285/350 105.0 330/421.5 16.2 2.49 16.14 6.48
7 225/346 276.5 285/349.5 110.9 330/421.5 16.5 2.49 16.73 6.71
8 225/344 262.2 285/349 107.9 340/424.5 18.1 2.43 14.48 5.96
9 225/344.5 289.1 280/350 117.9 340/424.5 15.3 2.45 18.86 7.69
10 225/349.5 302.9 285/352 132.4 350/435 14.7 2.29 20.55 8.98
11 225/351.5 253.9 285/354.5 117.9 340/432.5 14.2 2.15 17.88 8.30
a
Int.: intensity.
Table 3 Fluorescence spectral parameters of the sludge EPS samples at various EPS concentrations
Conc.
a
(mg C/l) Peak A Peak B Peak C A/B A/C B/C
Ex/Em Int.
b
Ex/Em Int.
b
Ex/Em Int.
b
AE-EPS 45.2 225/339 825.8 285/341.5 448.2 340/427.5 69.8 1.84 11.84 6.42
22.6 225/339 608.4 285/343.5 281.1 340/427 39.4 2.16 15.45 7.14
11.3 225/342.5 399.0 285/344 155.0 350/430.5 22.3 2.57 17.92 6.96
5.65 225/342 244.6 280/345 88.5 340/425.5 13.6 2.76 17.97 6.50
2.83 225/344.5 153.9 280/346 52.4 340/426 8.6 2.94 17.83 6.07
AN-EPS 29.6 225/347 527.7 285/351 382.0 340/432.5 47.6 1.38 11.08 8.02
14.8 225/347 474.9 285/350.5 249.3 330/427 27.7 1.90 17.12 8.99
7.4 225/346 331.7 285/349.5 138.5 340/426.5 17.1 2.39 19.36 8.09
3.7 225/346.5 208.8 285/350.5 76.3 330/424 11.1 2.74 18.74 6.85
1.85 225/344 132.4 285/349.5 44.2 330/426.5 6.6 3.00 19.92 6.64
a
Conc.: concentration.
b
Int.: intensity.
WAT E R R E S E A R C H 40 ( 2006) 1233 1239 1236
increased as pHwas increased from 3.0 to 7.0, but it decreased
as pH was further increased. On the other hand, for the AN-
EPS, these ratios increased as pH was increased from 3.0 to
10.0, and only slightly decreased thereafter. Such differences
conrm that the uorescence characteristics of the AE-EPS
and AN-EPS were not same.
3.3. Effect of EPS concentration on EEM uorescence
spectra
The uorescence characteristics of the EPS samples at various
EPS concentrations are listed in Table 3. The peak locations
were independent of the EPS concentration, but the peak
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Table 4 Fluorescence spectral parameters of the sludge EPS samples at various ionic strengths
Ionic strength (mM) Peak A Peak B Peak C A/B A/C B/C
Ex/Em Int.
a
Ex/Em Int.
a
Ex/Em Int.
a
AE-EPS 10 225/342.5 399.0 285/344 155.0 350/430.5 22.3 2.57 17.92 6.96
20 225/341.5 407.9 285/344 154.5 340/426 23.2 2.57 17.92 6.96
40 225/340 390.8 280/344 151.3 350/429.5 22.1 2.64 17.62 6.67
60 225/339.5 339.2 285/344 151.8 340/426 22.0 2.58 17.66 6.84
80 225/341 382.7 285/343.5 149.0 340/427 21.5 2.23 15.41 6.90
100 225/339 387.3 280/340.5 150.4 350/429 21.6 2.57 17.81 6.93
AN-EPS 10 225/346 331.7 285/349.5 138.5 340/426.5 17.1 2.39 19.36 8.09
20 225/341 290.9 285/347 113.0 330/423 15.6 2.39 19.36 8.09
40 225/340 323.0 285/345.5 126.1 330/426.5 14.3 2.57 18.70 7.26
60 225/340 321.1 280/345.5 126.3 330/424 15.6 2.56 22.63 8.84
80 225/341.5 337.3 285/344.5 131.6 330/426.5 14.0 2.54 20.56 8.09
100 225/339 342.3 280/345.5 141.6 330/426.5 14.7 2.56 24.18 9.43
a
Int.: intensity.
Fig. 2 EEM uorescence spectra of the AE-EPS at various pH values at an EPS concentration of 11.3mg C/l.
WATER RESEARCH 40 ( 2006) 1233 1239 1237
intensity depended heavily on the EPS concentration (Figs. 4c
and d). At a low EPS concentration (e.g., o10mg C/l), the peak
intensity had a linear relationship with the EPS concentra-
tion. On the other hand, at a high EPS concentration, the peak
intensity slowly increased with increasing EPS concentration,
but remained unchanged later. This might be attributed to the
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0
100
200
300
400
0
2 4 6 8 10 12 0 10 20 30 40 50 0 20 40 60 80 100
100
200
300
I
n
t
e
n
s
i
t
y
I
n
t
e
n
s
i
t
y
(a)
(b) (d) pH
0
200
400
600
800
0
200
400
600
(c)
Concentration (mg C / L)
0
100
200
300
400
0
100
200
300
(e)
(f) Ionic strength (mM)
Fig. 4 The intensities of uorescence peaks of: (a), (c), and (e) for AE-EPS; and (b), (d), and (f) for AN-EPS at various pH values,
EPS concentrations and ionic strengths.J: Peak A; K: Peak B; n Peak C.
Fig. 3 EEM uorescence spectra of the AN-EPS at various pH values at an EPS concentration of 7.4mg C/l.
WAT E R R E S E A R C H 40 ( 2006) 1233 1239 1238
inner ltering effect (Mobed et al., 1996). The peak intensity
ratios of Peak A to Peak B for both EPS spectra increased with
a decrease in EPS concentration. The intensity ratios of Peak A
to Peak C for both EPS initially increased with a decrease in
EPS concentration, but later remained almost unchanged.
However, the ratios of Peak B to Peak C remained constant.
The difference in the peak intensity ratio might be attributed
to the low concentration of humic substances in the EPS.
3.4. EEM spectra of EPS at various ionic strengths
Table 4 shows EEM uorescence spectral characteristics of the
EPS at various ionic strengths. The peak locations, peak
intensities and the ratios of different peaks in EEM spectra of
the EPS samples were not substantially inuenced by ionic
strength (Figs. 4e and f). These results agree well with those of
Mobed et al. (1996). They observed that the ionic strength
(01.0M KCl) had no signicant inuence on EEM spectra of
aquatic humic samples or the soil-derived humic acids.
4. Conclusions
As one of the rapid, sensitive and selective analytical
methods, EEM uorescence spectroscopy was found to be
appropriate for characterizing the EPS extracted from the
aerobic and anaerobic sludge in wastewater treatment
systems. Three individual uorescence peaks (Peak A at 225/
340350nm, Peak B at 280285/340350nm, Peak C at 330340/
420430nm) were identied in EEM uorescence spectra for
the sludge EPS samples. Peaks A and B were attributed to the
protein-like uorophores, and Peak C to the humic-like
uorophore. Both pH and EPS concentration had signicant
effects on EEM uorescence spectra of the sludge EPS, but the
ionic strength had no substantial inuence on the spectra.
The differences in the uorescence parameters of the sludge
EPS indicated the difference in the chemical structures of EPS
from various origins. EEM spectroscopy was proven to be an
appropriate and effective method to characterize the EPS
from various origins in wastewater treatment systems.
Acknowledgments
The authors wish to thank the Natural Science Foundation of
China (NSFC) (Grant No. 20577048), and the NSFC-RGC Joint
Project (50418009) for the partial support of this study.
R E F E R E N C E S
APHA, 1995. Standard Methods for the Examination of Water and
Wastewater, 19th ed. American Public Health Association,
New York.
Baker, A., 2001. Fluorescence excitationemission matrix char-
acterization of some sewage-impacted rivers. Environ. Sci.
Technol. 35, 948953.
Baker, A., Inverarity, R., 2004. Protein-like uorescence intensity
as a possible tool for determining river water quality. Hydrol.
Process. 18, 29272945.
Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003.
Fluorescence excitationemission matrix regional integration
to quantify spectra for dissolved organic matter. Environ. Sci.
Technol. 37, 57015710.
Coble, P.G., 1996. Characterization of marine and terrestrial DOM
in seawater using excitationemission matrix spectroscopy.
Mar. Chem. 51 (4), 325346.
Esparza-Soto, M., Westerhoff, P.K., 2001. Fluorescence spectro-
scopy and molecular weight distribution of extracellular
polymers from full-scale activated sludge biomass. Water Sci.
Technol. 43 (6), 8795.
Frolund, B., Palmgren, R., Keiding, K., Nielsen, P.H., 1996. Extrac-
tion of extracellular polymers from activated sludge using a
cation exchange resin. Water Res. 30, 17491758.
Her, N., Amy, G., McKnight, D., Sohn, J., Yoon, Y., 2003.
Characterization of DOM as a function of MW by uores-
cence EEM and HPLC-SEC using UVA, DOC, and uorescence
detection. Water Res. 37, 42954303.
Lu, X.Q., Jaffe, R., 2001. Interaction between Hg(II) and natural
dissolved organic matter: a uorescence spectroscopy based
study. Water Res. 35, 17931803.
Mobed, J.J., Hemmingsen, S.L., Autry, J.L., McGown, L.B., 1996.
Fluorescence characterization of IHSS humic substances: total
luminescence spectra with absorbance correction. Environ.
Sci. Technol. 30, 30613065.
Patel-Sorrentino, N., Mounier, S., Benaim, J.Y., 2002. Excitation-
emission uorescence matrix to study pH inuence on organic
matter uorescence in the Amazon basin rivers. Water Res. 36,
25712581.
Peuravuori, J., Koivikko, R., Pihlaja, K., 2002. Characterization,
differentiation and classication of aquatic humic matter
separated with different sorbents: synchronous scanning
uorescence spectroscopy. Water Res. 36, 45524562.
Pullin, M.J., Cabaniss, S.E., 1995. Rank analysis of the
pH-dependent synchronous uorescence spectra of six
standard humic substances. Environ. Sci. Technol. 29,
14601467.
Reynolds, D.M., 2002. The differentiation of biodegradable and
non-biodegradable dissolved organic matter in waste waters
using uorescence spectroscopy. J. Chem. Technol. Biotechnol.
77, 965972.
Reynolds, D.M., Ahmad, S.R., 1995. The effect of metal ions
on the uorescence of sewage waste water. Water Res. 29,
22142216.
Sheng, G.P., Yu, H.Q., Yu, Z., 2005. Extraction of the extracellular
polymeric substances from a photosynthetic bacterium Rho-
dopseudomonas acidophila. Appl. Microbiol. Biotechnol. 67,
125130.
Staudt, C., Horn, H., Hempel, D.C., Neu, T.R., 2004. Volumetric
measurements of bacterial cells and extracellular polymeric
substance glycoconjugates in biolms. Biotechnol. Bioeng. 88,
585592.
Wilen, B.M., Jin, B., Lant, P., 2003. The inuence of key chemical
constituents in activated sludge on surface and occulating
properties. Water Res. 37, 21272139.
Wingender, J., Neu, T.R., Flemming, H.C., 1999. Microbial Extra-
cellular Polymeric Substances: Characterization, Structures
and Function. Springer, Berlin, Heidelberg.
Yamashita, Y., Tanoue, E., 2003. Chemical characterization of
protein-like uorophores in DOM in relation to aromatic
amino acids. Mar. Chem. 82, 255271.
ARTICLE IN PRESS
WATER RESEARCH 40 ( 2006) 1233 1239 1239