Comparison of the characteristics of extracellular polymeric substances for two different extraction methods and sludge formation conditions

Chemosphere 90 (2013) 237–244

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Comparison of the characteristics of extracellular polymeric substances for two different extraction methods and sludge formation conditions Bo-Mi Lee a, Hyun-Sang Shin b, Jin Hur a,⇑ a b

Department of Environment and Energy, Sejong University, Seoul 143-747, South Korea Department of Environmental Engineering, Seoul National University of Science and Technology, Seoul 200-701, South Korea

h i g h l i g h t s " The EPS using CER contain more aromatic condensed structures with less acidic functional groups. " Hg(II) binding was measurable only for the EPS using formaldehyde/NaOH. " The highest pyrene Koc value was obtained for the EPS of anaerobic sludge using CER. " The extraction methods produced more differences in the EPS characteristics than the sludge formation conditions.

a r t i c l e

i n f o

Article history: Received 7 May 2012 Received in revised form 26 June 2012 Accepted 30 June 2012 Available online 24 July 2012 Keywords: Anaerobic Fluorescence Size exclusion chromatography Fourier transform infrared spectroscopy Binding affinity

a b s t r a c t The characteristics of extracellular polymeric substances (EPSs) were compared for two different extraction methods and dissimilar sludge formation conditions (aerobic versus anaerobic). The measured characteristics included specific ultraviolet absorbance (SUVA) values, fluorescence excitation–emission matrices, molecular weight distributions, Fourier transform infrared (FT-IR) spectra, and the binding affinities for pyrene and Hg(II). The analyses demonstrated that the EPS extracted using cation exchange resin (CER) were composed of more aromatic and more condensed structures with higher molecular weight than those using formaldehyde/NaOH. The FT-IR results further revealed that the EPS using CER contained relatively lower content of protein to carbohydrate and less acidic functional groups (i.e., ACOOH or AOH groups). The observed differences between the two extraction methods were more pronounced for the EPS of anaerobic sludge compared to those of aerobic sludge. The extent of pyrene binding and the apparent stability constants for Hg(II) were very consistent with the implications from the previous EPS physicochemical characteristics. The highest extent of pyrene binding was observed for the EPS of anaerobic sludge using CER while no measurable Hg(II) stability constant was found for the same EPS sample. Our results demonstrated that the EPS characteristics including their binding affinities are likely strongly affected by the sludge formation conditions as well as the extraction methods although the latter produced more differences. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Extracellular polymeric substances (EPSs), present outside of cells as well as in the interior of microbial aggregates, are excreted by microorganisms in many biological operation systems such as sludge-based wastewater treatment processes (Liu and Fang, 2002). EPS are composed of various constituents such as polysaccharides, proteins, uronic acids, humic-like substances, lipids, and DNA (Sheng et al., 2010). It is reported that the structures and the characteristics of EPS may influence the adhesion and the settling properties of sludge, and the extent of membrane fouling ⇑ Corresponding author. Tel.: +82 2 3408 3826; fax: +82 2 3408 4320. E-mail address: [email protected] (J. Hur). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.06.060

(Chang et al., 2002; Liu and Fang, 2003; Ahmed et al., 2007). EPS can also bind with heavy metals and organic pollutants because they retain both hydrophobic and hydrophilic regions within their structures (Sheng et al., 2008). Recently, the binding affinities of EPS were highlighted in relation to the ability of sewage sludge to remove organic and inorganic pollutants (Pan et al., 2010; Zhang et al., 2010). The composition of EPS is known to differ by the redox state upon the formation of the sludge (i.e., aerobic and anaerobic conditions). During anaerobic digestion, for example, protein and carbohydrate compounds within EPS tend to be more degraded while humic-like substances may be enriched (Nielsen et al., 1996). It has also been reported that a higher ratio of protein to carbohydrate for the EPS is extracted from anaerobially digested sludge compared to

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the raw sludge formed under aerobic condition (Houghton et al., 2000). The fluorescence characteristics of EPS such as the locations and the intensities of peaks and the peak ratios may vary with different sludge formation conditions (Sheng and Yu, 2006; Sheng et al., 2008). There are many extraction methods proposed to obtain EPS from raw sludge samples, which include sonication, high speed centrifugation, sonication/centrifugation, heating, alkaline, sulfide, EDTA, CER, and formaldehyde/NaOH treatment (Liu and Fang, 2003; Sheng et al., 2010). The type of the extraction method used may result in the variations of the quantity of the EPS and the characteristics. Domínguez et al. (2010) have demonstrated that the recovery rates and the fluorescence characteristics of EPS varied with the types of the extraction methods. Among the pool of the EPS extraction methods suggested, CER and formaldehyde/NaOH methods have been the most widely used for the EPS extraction. Although the formaldehyde/NaOH method is known to produce a higher recovery rate than the CER method, the latter is preferred when a lower extent of cell lysis and the minimum amount of by-product pollutants are required (Sheng et al., 2010). To date, a number of analytical techniques have been applied for characterization of EPS, which include fluorescence spectroscopy, size exclusion chromatography (SEC), Fourier transform infrared spectroscopy (FT-IR), gas chromatography, 13C nuclear magnetic resonance (13C NMR), and so on (Sheng et al., 2010). Among those, the first three analyses have been the most frequently used for the EPS characterization due to the relatively less requirement of the sample amount and the pretreatment procedures. Ni et al. (2009) have utilized the fluorescence excitation– emission matrix (EEM) to observe the changes in EPS composition during the sludge cultivation process. SEC has been used for comparing the molecular size distribution of EPS under various environmental conditions (Nielsen et al., 1996; Comte et al., 2007). From the FT-IR absorption peaks, the major components of EPS can be qualitatively distinguished such as proteins (amino acids), fats (aliphatic ester), polysaccharides (ether), and other organics (Ramesh et al., 2006). Despite the recent advances in characterizing EPS, more knowledge is still required for fully understanding of the differences among the characteristics of the EPS that are produced under different conditions. It has long been a neglected topic whether or not the binding affinities of EPS with organic and inorganic pollutants may vary with different extraction methods and different sludge formation conditions. In particular, to the authors’ knowledge, this is the first report on comparing the interactions between a hydrophobic organic contaminant and the EPS that are produced under different conditions. Therefore, the main objective of this study was to compare the characteristics of the EPS including their binding affinities for two representative extraction methods and two different sludge formation conditions. The major EPS characterization included here are SUVA, fluorescence EEM, SEC, and FT-IR. Pyrene and Hg(II) binding properties were also determined by examining the interactions of EPS with organic and inorganic pollutants. 2. Materials and methods 2.1. Sludge sample collection and EPS extraction Aerobic sludge was collected from an aeration tank in a municipal wastewater treatment plant, located in the city of Uijeongbu, Korea. The facility has a treatment capacity of 153 000 m3 d1 and it adopts an advanced biological treatment process, namely, the MLE (Modified Ludzack Ettinger) process. The process commonly used for the simultaneous removal of organics and nitrogen in an activated sludge-based wastewater treatment system (Liang

et al., 2010). MLE consists of anoxic, aerobic basin, and a secondary clarifier and it internally recycles nitrate-enriched mixed liquor suspended solids (MLSSs) from aerobic reactors to anoxic basins. Anaerobic sludge was collected after the anaerobic tank in the same wastewater treatment facility. Both collected sludges were stored under 4 °C and transported to a laboratory. Total suspended solids (TSSs) and volatile suspended solids (VSSs) were measured immediately in the laboratory. The ratios of VSS/TSS were 62.4% and 57.4% for aerobic and anaerobic sludges, respectively. EPS were extracted by the two methods using formaldehyde/ NaOH and CER, generally following the methodologies suggested by Domínguez et al. (2010). For the formaldehyde/NaOH method, 50 mL of the original sludge was centrifuged at 5000 rpm for 15 min at 4 °C. The sludge pellets were re-suspended in 50 mL of 0.05% NaCl solution. A small amount (0.3 mL) of 37% formaldehyde was then added in the sludge/NaCl solution. The solution was stirred for 1 h at 900 rpm under 4 °C. Afterwards, 20 mL of 1 N NaOH was added and it was stirred again for 3 h under the same condition. The solution was finally centrifuged at 5000 rpm for 15 min and the supernatant was filtrated with 0.45 lm cellulose acetate membrane filter (Advantec, Japan). For the CER method, 150 mL of sludge was centrifuged at 5000 rpm for 15 min at 4 °C. The sludge was re-suspended in 150 mL of a buffer solution (pH 7.0) containing 2 mM Na3PO4, 4 mM NaH2PO4, 9 mM NaC1, and 1 mM KCI (Frølund et al., 1996). Cation exchange resin (Dowex 50 W  8–100, Sigma–Aldrich, USA) was then used for the re-suspended solution with a constant ratio of 70 g CER g VSS1 before the solution was stirred at 900 rpm under 4 °C for 16 h. CER was first taken out by gravity separation and the remaining sludge and CER were both finally removed by centrifugation at 5000 rpm for 15 min at 4 °C. Aliquots of the extracted EPS were freeze dried for FT-IR analyses. 2.2. Dissolved organic carbon (DOC) and UV–visible absorbance measurements DOC concentrations of the extracted EPS were measured using a TOC analyzer (Shimadzu V-series, TOC-CHP). A full scan of ultraviolet (UV)–visible spectra of the EPS was made by employing a UV– visible spectrophotometer (HACH, DR 5000). Absorbance spectral slopes between 275 nm and 295 nm were calculated using linear regression of the log-transformed spectra (Helms et al., 2008). SUVA values of the EPS were determined by dividing 100-fold of the UV absorbance at 254 nm by the DOC concentration. DOC concentrations and pH values of the EPS samples were adjusted to be 5 mg C L1 and 3.0, respectively, before the UV absorbance and the fluorescence measurements. 2.3. Fluorescence measurements Fluorescence spectra were measured using a luminescence spectrometry (LS-50B, Perkin-Elmer). Excitation and emission slits were both adjusted at 10 nm. A 290 nm cut-off filter was used to limit second-order Raleigh light scattering. The original fluorescence spectra were adjusted by inner-filter correction (Gauthier et al., 1986; Hur et al., 2009). The fluorescence response to a blank solution (Milli-Q water) was subtracted from the fluorescence of each sample. Humification index (HIX) was estimated using a sum of the emission intensities from 485 nm to 550 nm at an excitation wavelength of 465 nm (Milori et al., 2002). A higher HIX value is typically associated with the presence of more aromatic structures and/or a high degree of conjugation unsaturated aliphatic chains (Fuentes et al., 2006). Fluorescence index (FI) was calculated using a ratio of emission intensities at the wavelength of 450–500 nm with a fixed excitation wavelength of 370 nm (McKnight et al.,

B.-M. Lee et al. / Chemosphere 90 (2013) 237–244

2001). FI has been used to distinguish between microbial and terrestrial origins of dissolved organic matter (DOM). A high FI value close to 2.0 indicates that the corresponding DOM may originate from microbially produced materials. EEMs were measured at the excitation wavelengths from 220 to 500 nm with 5 nm increments, and at the emission wavelengths between 280 and 550 nm with 0.5 nm increments. The scanning speed was set at 1200 nm min1. The same pH value of the samples as UV absorbance measurements (i.e., pH 3.0) was maintained for all the fluorescence analyses. 2.4. Molecular weight distribution measurements Molecular weight (MW) distributions of the samples were determined by employing a SEC system, consisting of a high performance liquid chromatography (HPLC, Waters), a UV detector (UV–visible 2489, Waters), and a protein-Pak 125 column (Waters) with a separation MW range of 2–80 kDa. The mobile phase was composed of 0.002 M NaH2PO4, 0.002 M Na2HPO4, and 0.1 M NaCl to maintain a pH of 6.8. All standards (Sodium polystyrene sulfonate 18 K, 8 K, 5.4 K, 1.8 K, acetone) and the samples were measured at a detection wavelength of 254 nm. The flow rate of the mobile phase was maintained at 1 mL min1. Weight- and number–average molecular weight (MWw and MWn) values were calculated following Hur and Schlautman (2003). Polydispersity was estimated using a ratio of MWw to MWn values. Because of the limitation of the detectable molecular size of the column used, the SEC chromatograms generated for this study were utilized in order to operationally differentiate among the EPS samples.

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where [Pyr-DOM] is the concentration of pyrene that is involved in binding with DOM (lg L1), [Pyr]free is the concentration of freely dissolved pyrene (lg L1), and [DOM] is 15 mg C L1. Concentration of [Pyr]free was calculated by subtracting [Pyr-DOM] from the initial pyrene concentration. To perform the Hg (II) binding experiment, the DOC concentration and the pH value of each EPS sample were first adjusted to be 10 mg C L1 and 9.0, respectively. Appropriate amounts of HgCl2 stock solutions (0.005, 0.001, 0.005 M) were spiked into 1 mL of EPS solutions contained in polypropylene vials (2 mL) so that the final Hg (II) concentrations in the vials ranged from 0 to 150 lM (0, 5, 10, 20, 30, 50, 75, 100 and 150 lM). The vials were then shaken for 30 min at 200 rpm in room temperature to equilibrate (Hur and Lee, 2011). Fluorescence intensities were then measured at the locations of the EEM peaks. The stability constants (KM) and the fraction of the initial fluorescence involving binding sites (f) were calculated using a modified Stern–Volmer Eq. (2) (Hur and Lee, 2011).

F0 1 1 ¼ þ F 0  F ðf  K M  C M Þ f

ð2Þ

where F0 and F are the initial fluorescence intensity with no metal added and the measured fluorescence intensities after the addition of metal concentration (CM), respectively.

3. Results

2.5. FT-IR measurements

3.1. The extraction rates and the spectroscopic properties of EPS

The FT-IR spectra of the EPS samples were recorded using an FTIR spectrometer (Smiths, Travel IR). The freeze dried EPS samples were initially mixed with KBr (1 mg of EPS per 100 mg of KBr). The KBr pellets (FT-IR grade, Aldrich) were then dried by heating and they were kept under vacuum in a desiccator prior to use. The blank was corrected using a clean KBr pellet. The spectra were obtained with a resolution of 4 cm1 and a scan range of 600– 4000 cm1.

Much higher extraction rates as a basis of DOC concentration per mass of VSS were observed for the formaldehyde/NaOH method compared to the CER method although the EPS of aerobic sludge versus anaerobic sludge consistently exhibited higher extraction rates irrespective of the extraction types (Table 1). The extraction method using formaldehyde/NaOH appears to be very efficient in producing EPS, overwhelming the influence of the sludge formation conditions. Our results of the extraction efficiencies agreed well with prior studies. For example, Liu and Fang (2002) and Comte et al. (2007) reported the highest extraction rate of the formaldehyde/NaOH method among several extraction methods tested. Lei et al. (2007) observed a decreasing trend in the quantities of the EPS in sludge during anaerobic digestion. However, the possibility cannot be ruled out that potential contamination from formaldehyde may partially contribute to the high extraction rate (D’Abzac et al., 2010b). SUVA values ranged from 0.69 to 2.81 L mg C1 m1 with higher values for the EPS using CER. However, no consistent difference in the SUVA values was observed between the EPS of aerobic and anaerobic sludges for the two different methods. A higher SUVA value was exhibited for the EPS of aerobic sludge extracted using formaldehyde/NaOH whereas the opposite trend was shown for the EPS using CER (Table 1). S275–295 values tended to be higher for the EPS of aerobic versus anaerobic sludges independent of the extraction methods. Higher S275–295 values may be associated with a lower molecular size of DOM (Helms et al., 2008). The FI values close to 2.0 were exhibited for the EPS using CER, suggesting that the CER method may be more appropriate in distinguishing EPS, which is microbial-derived DOM, from other sources (e.g., terrestrial) of DOM. Similarly to the SUVA values, higher HIX was observed for the EPS using CER versus formaldehyde/NaOH (Table 1). A substantial difference in the HIX values between aerobic and anaerobic sludge was only found for the EPS using CER.

2.6. Pyrene/Hg (II)-EPS binding experiments The fluorescence quenching technique was used to quantify the binding affinities of EPS with pyrene and Hg (II). Pyrene-EPS binding experiments were conducted following a methodology suggested by Hur et al. (2009). Pyrene (Sigma–Aldrich, 99.9%) has often been used as a model hydrophobic organic contaminant (HOC) for investigating the interactions between DOM and HOC. To obtain pyrene-EPS binding coefficients (Koc), EPS solutions were prepared at a constant concentration of 15 mg C L1 and the ion strength and pH were adjusted as 0.1 M and 7.0, respectively, using NaCl and 0.1 N HCl. An aliquot of pyrene stock solution in methanol was spiked into each EPS solution so that the final concentration of pyrene in the samples was 12 lg L1. The solutions were then shaken enough to reach an equilibrium condition for 15 min. Inner-filter correction was made to account for light absorption by the presence of DOM (Gauthier et al., 1986). Fluorescence intensities of samples were measured at excitation/emission wavelengths of 336/373 nm. The concentration of freely dissolved pyrene in each EPS solution was quantified from a standard curve based on pure pyrene solution with five different concentrations. The Koc values were calculated by the following equation:

K oc ¼

½Pyr-DOM ½Pyrfree ½DOM

ð1Þ

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Table 1 Extraction rates and selected spectroscopic indices of the four different EPS samples.

a b c

Extraction methods

Sludge type

VSS (mg C g1)

SUVA (L mg C1 m1)

S275–295a

FIb

HIXc

Formaldehyde/NaOH

Aerobic Anaerobic

134.6 67.3

0.94 0.69

0.023 0.018

1.63 1.70

11.15 11.74

CER

Aerobic Anaerobic

13.6 7.6

2.00 2.81

0.036 0.021

1.87 2.07

16.13 40.20

S275–295: Spectral slope based on log-transform from 275 nm to 295 nm (Helms et al., 2008). FI: Fluorescence index (McKnight et al., 2001). HIX: Humification index (Milori et al., 2002).

3.2. Fluorescence EEM spectra The measured EEM spectra are presented in Fig. 1. The classification of the peaks in the EEM spectra was based on Hudson et al. (2008) and the detailed peak locations and the intensities are described in Table 2. Three peaks (Peaks T1, T2, and C) were commonly observed for all the EPS samples. The locations of the three peaks were similar to those reported in other studies using EPS (Domínguez et al., 2010; Ramesh et al., 2006; Ni et al., 2009). The two peaks, located near 220 nm/340–360 nm and 275 nm/ 350 nm (excitation/emission wavelengths), were both assigned to

tryptophan-like fluorescence peak, named as Peak T1 and Peak T2, respectively. The peak (Peak C) appearing at the wavelengths of 330 nm/415–430 nm is typically defined as humic-like fluorescence peak. Peak C was placed at a longer emission wavelength for the EPS using CER compared to those using formaldehyde/NaOH while no difference in the location was observed between the EPS of aerobic and anaerobic sludges extracted by the same method. The orders of the designated peak intensities per organic carbon were consistently T1 > T2 > C irrespective of the extraction types and the sludge formation conditions. However, mixed results have been

500

500

(a) Peak C

400

350

450

Excitation (nm)

Excitation (nm)

450

Peak T2 Peak T1

400

300

250

250

350

400

450

500

Peak A

350

300

300

(b)

550

300

350

Emission (nm) 500

450

500

550

500

550

500

(c)

450

400

Excitation (nm)

Excitation (nm)

450

400

Emission (nm)

350

400

350

300

300

250

250

300

350

400

450

Emission (nm)

500

550

(d)

300

350

400

450

Emission (nm)

Fig. 1. Fluorescence EEM spectra of the EPS of: (a) aerobic and (b) anaerobic sludges using the formaldehyde/NaOH method, and of (c) aerobic and (d) anaerobic sludges using the CER method. Peaks T1 and T2 represent trytophan-like fluorescnece peaks. Peak C and Peak A indicate humic-like and fulvic-like fluorescence peaks, respectively.

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B.-M. Lee et al. / Chemosphere 90 (2013) 237–244 Table 2 The peak locations (excitation/emission) and the intensities of the fluorescence EEM spectra for the four EPS samples. Extraction methods

*

Sludge type

Peak T1

Peak T2 *

Peak C

Peak A

Locations

Int.

Locations

Int.

Locations

Int.

Locations

Int.

Formaldehyde/NaOH

Aerobic Anaerobic

220/360 220/360

278.7 317.6

275/352 280/352

92.0 92.0

330/420 335/420

47.9 47.9

– 270/439

– 112.6

CER

Aerobic Anaerobic

220/351 220/339

379.3 248.4

275/356 280/349

145.3 81.2

335/428 330/427

24.3 30.8

– 275/430

– 49.1

Int.: Fluorescence intensity based on a constant DOC concentration of the samples.

Table 3 The relative ratios of the four fluorescence peaks for the EPS samples. Extraction methods

Sludge type

T1/T2

T1/C

T1/A

T2/C

C/A

Formaldehyde/NaOH

Aerobic Anaerobic

3.03 3.45

5.82 6.63

– 2.82

1.92 1.92

– 0.40

CER

Aerobic Anaerobic

2.61 3.06

15.58 8.07

– 5.06

5.97 2.64

– 0.60

reported on the relative fluorescence peak intensities of EPS. For example, the same trend as our study was reported for the EEM peak intensities in a study by Ramesh et al. (2006) using various types of sludges whereas Domínguez et al. (2010) have shown that the orders of the peak intensities depended on the types of the extraction methods. A fulvic-like fluorescence peak (Peak A), placed near the wavelengths of 270 nm/435 nm, was observed only for the EPS of anaerobic sludge, suggesting that the peak may be used as an EEM index to discriminate between the EPS of aerobic and anaerobic sludge irrespective of the two extraction methods. The peak ratios of T1/T2 were similar among the samples, ranging from 2.61 to 3.06, indicating that the relative distribution of the two peaks may not be affected by the sludge formation condition as well as the EPS extraction methods (Table 3). However, higher

T1/C ratios were observed for the EPS using CER than those using formaldehyde/NaOH. For the CER method, the EPS of aerobic sludge exhibited a much higher ratio of T1/C than that of anaerobic sludge whereas such a difference between the two EPS was not obvious for the formaldehyde/NaOH method. The observed relative abundance of Peak T1 and Peak C was supported by some indirect evidence of other prior studies. For example, the presence of humic substances (HSs) tends to be more pronounced during anaerobic digestion (Nielsen et al., 1996). Liu and Fang (2002) demonstrated higher concentrations of HS in the EPS using formaldehyde/NaOH versus CER. Similar trends were observed for the other fluorescence peak ratios (i.e., T1/A and T2/C) (Table 3) although the differences in the peak ratios between the two sludge formation conditions were only observed for EPS using CER. In addition, a higher ratio of Peak C/Peak A was observed for the EPS using CER versus formaldehyde/ NaOH. The overall fluorescence EEM patterns of the EPS using CER agreed well with the results of Sheng and Yu (2006) using the same extraction method. 3.3. Molecular weight distributions Three peaks were shown in the SEC chromatograms of the EPS using formaldehyde/NaOH. Each of the peaks corresponded to the MW values of 600–700 Da, 1350–1570 Da, and over 30 000 Da 1.2

1.2 1.0

(a)

MW w = 5285 Da MW n = 926 Da Polydispersity = 5.71

1.0

Signal

0.6

0.6

0.4

0.4

0.2

0.2

0.0 10

100

1000

10000

0.0 10

100000

100

Molecular Weight (Da) 1.2

(c)

MW w = 5418 Da MW n = 265 Da Polydispersity = 20.4

1.0

Signal

Signal

10000

100000

MW w = 9305 Da MW n = 431 Da Polydispersity = 21.6

(d)

0.8

0.8 0.6

0.6

0.4

0.4

0.2

0.2

0.0 10

1000

Molecular Weight (Da)

1.2 1.0

MW w = 6349 Da MW n = 707 Da Polydispersity = 8.98

0.8

0.8

Signal

(b)

100

1000

10000

Molecular Weight (Da)

100000

0.0 10

100

1000

10000

100000

Molecular Weight (Da)

Fig. 2. Size exclusion chromatograms of the EPS of: (a) aerobic and (b) anaerobic sludges based on the formaldehyde/NaOH method, and of (c) aerobic and (d) anaerobic sludges based on the CER method.

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B.-M. Lee et al. / Chemosphere 90 (2013) 237–244 Table 4 General assignments of the main peaks observed for the FT-IR spectra of the EPS samples (Guibaud et al., 2003; Ramesh et al., 2006; D’Abzac et al., 2010b).

(a)

Wavenumber (cm1)

Assignments

3300 2930 1600–1650 1400

Stretching of ANH2 or AOH groups Stretching of CAH of lipid C@O and CAN stretching of amide I C@O stretching of COO and deformation stretching of OH (alcohols and phenols) and/or NACAH (protein) CAO stretching of polysaccharide

Absorbance (arb. unit)

1050

(b)

(c)

(d) 4000

3500

3000

2500

2000

1500

1000

-1

Wave Number (cm ) Fig. 3. The FT-IR spectra of the EPS of: (a) aerobic and (b) anaerobic sludges based on the formaldehyde/NaOH method, and of (c) aerobic and (d) anaerobic sludges based on the CER method.

(Fig. 2). The EPS of anaerobic sludge exhibited a higher signal toward larger molecular sizes compared to those of aerobic sludge. The SEC chromatograms of the EPS using CER were different from those using formaldehyde/NaOH in that they had an additional peak in the MW range of <300 and more pronounced peaks in higher MW ranges (Fig. 2). Domínguez et al. (2010) also observed a greater number of peaks in the SEC chromatograms for the EPS using CER than those using formaldehyde/NaOH. The MWw values of the four EPS samples reflected well their SEC chromatograms, exhibiting higher values for the EPS of anaerobic versus aerobic sludge and/or for the EPS using CER versus formaldehyde/NaOH method. However, the MWn values were not consistent with the order expected from the apparent chromatograms. Much higher polydispersity values of the EPS using CER versus formaldehyde/NaOH may indicate that the EPS extracted by the CER method tend to be composed of more various sized components. 3.4. FT-IR spectra of EPS Fig. 3 shows the FT-IR spectra of the four EPS samples. The spectra agreed well with the absorption bands typically observed for other EPS studies (Guibaud et al., 2003; Ramesh et al., 2006). The strong bands were evidently shown in the regions of 3300 cm1, 1600–1650 cm1, 1400 cm1, and 1050 cm1. On the other hand, the bands at 2930 cm1 were relatively very weak. The absorption

bands at 1250 cm1, which is associated with deformation vibration of C@O of nucleic acids, were not observed in the spectra. The overall FTIR results indicate that protein and carbohydrate constitute the main structures of the four EPS samples (Table 4), which is in line with other literature evidencing that EPS from activated sludge are composed of protein and carbohydrate as major constituents, and humic-like substances, lipid and nucleic acids in smaller quantities (Guibaud et al., 2003; Comte et al., 2006). Although the positions of the absorbance bands for protein and carbohydrate appeared similar for all the EPS samples, the relative peak intensities, which are associated with the relative amount of each functional group, differed by the samples. For example, the EPS using formaldehyde/NaOH exhibited relatively strong bands around 1600 cm1 but weak bands at 1050 cm1 whereas the opposite trend was found for the EPS using CER. This result suggests that the content of protein relative to carbohydrates may be higher for the EPS using formaldehyde/NaOH than those using CER. The peaks at 3300 cm1 and 1400 cm1 were more prominently present for the EPS using formaldehyde/NaOH, implying more abundance of acidic functional groups (i.e., AOH, ACOOH) compared to those using CER. Subtle differences in the spectra were observed between the EPS of aerobic and anaerobic sludge as well. For the EPS of anaerobic sludge using CER, the band around 1630 cm1 was slightly separated into two weak bands at 1630 cm1 and 1580 cm1, which corresponds to the presence of amide II (combination of CAH bending and NAH stretching). In addition, two distinct bands at 850 and 950 cm1 were shown only for the EPS of anaerobic sludge, which may be attributed to several visible bands related to phosphate or sulfur functional groups (Comte et al., 2006) and/or the occurrence of possible linkages between two monosaccharide molecules (Parikh and Madamwar, 2006). 3.5. Pyrene and Hg binding properties Pyrene-EPS binding coefficients (Koc) ranged from 12.0 to 26.0 L g1 for the four different EPS. The highest Koc value was observed for the EPS of anaerobic sludge using CER (Table 5). The Koc values reported here were lower than those of DOM using different sources such as aquatic humic substances, soil and sediment (Koc > 40). The difference may be attributed to the dominant presence of the organic components not involving the binding in the EPS such as proteins and polysaccharides, which are associated with high polarity and low aromatic content (Wicke et al., 2007). Hg(II) binding properties of the EPS samples were compared using the model parameters calculated at three prominent peaks in the EEMs (Peak T1, T2 and C). All the titration data appear to fit well with our suggested fluorescence quenching model as indicated by the high correlation coefficients (r > 0.99) and the statistical significances (p values < 0.001). The estimated stability constants expressed by log KM values ranged from 4.2 to 4.7 at the selected peaks among the samples. The ranges of the values were comparable to those of a prior report using other types of

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B.-M. Lee et al. / Chemosphere 90 (2013) 237–244 Table 5 Pyrene and Hg(II) binding properties of the four EPS samples. Extraction methods

Sludge types

Pyrene-binding Koc (L g

1

)

Hg(II)-bindinga Peak wavelength (ex./em.)

r

Log KM

f

225/355 280/357 320/413 225/362 280/359 320/409

0.994b 0.990b 0.993b 0.996b 0.994b 0.993b

4.67 ± 0.02 4.67 4.33 4.46 4.58 4.43

0.71 ± 0.04b 0.74 0.58 0.74 0.64 0.68

225/360 280/358 335/434 225/354 275/354 335/438

0.990b – – – – –

4.16 – – – – –

0.81 – – – – –

Formaldehyde/NaOH

CER

Aerobic

13.9 ± 1.6

Anaerobic

12.0 ± 1.6

Aerobic

13.4 ± 2.3

Anaerobic

26.0 ± 1.7

a Correlation coefficients of predicted versus observed fluorescence intensity (r), conditional stability constants (Log KM), and fraction of the initial fluorescence corresponding to binding sites (f). b p < 0.001.

EPS (Zhang et al., 2010). Except for Peak T1 of the EPS of aerobic sludge, no fluorescence quenching at the selected peaks was observed upon the addition of Hg(II) for the EPS using CER. Furthermore, the Hg(II) binding observed at Peak T1 was even much weaker than those of the EPS using formaldehyde/NaOH. In contrast to the stability constants, the f values of the EPS of aerobic and anaerobic sludge were nearly indistinguishable, each ranging from 0.58 to 0.74 and from 0.64 to 0.74 respectively (Table 4). 4. Discussion The SUVA and the fluorescence properties (i.e., HIX and FI values) of our EPS samples suggest that the CER method extracted the EPS containing more aromatic carbon structures from the original sludge compared to the formaldehyde/NaOH method. The presence of more condensed organic structures for the EPS using CER versus formaldehyde/NaOH was also supported by the location of Peak C at a longer emission wavelength (i.e., red-shifting) for the fluorescence EEM data (Fuentes et al., 2006). Peak A may be suggested as an indicator to discriminate between the EPS of aerobic and anaerobic sludge irrespective of the two extraction methods due to the presence of the peak only for the EPS of anaerobic sludge. In general, the differences in fluorescence EEM patterns between the EPS of aerobic and anaerobic sludge were more pronounced for the EPS extracted by the CER method. Comparison of the SEC chromatograms revealed that relatively large sized UV-absorbing components were distributed in the EPS of anaerobic versus aerobic sludge. Nielsen et al. (1996) demonstrated, based on their SEC results, that smaller sized organic compounds of sludge were easily degraded during anaerobic digestion whereas high molecular weight components tended to remain in the process. Our combined SEC results are very consistent with the prior report as well as the implications obtained from SUVA, fluorescence EEM, and other fluorescence indices for this study. Our Koc results also agreed with the previous results of the values of SUVA, fluorescence indices, and MWw. It is evident from lots of literature that a higher extent of pyrene binding is associated with higher SUVA, HIX, and MWw values of DOM (Chin et al., 1997; Hur and Kim, 2009). No quenching with Hg(II) addition for most fluorescence peak locations of the EPS using CER indicates that the CER method may not be suitable for studying the binding properties of EPS with heavy metals. The observed differences in the Hg(II) binding properties between the EPS using the two extraction methods were explained well by our previous FT-IR spectra, in which the EPS using formaldehyde/NaOH exhibited more pronounced absorption bands

associated with metal binding (i.e., ACOOH and AOH groups). The comparison for the binding properties between the two different methods also agrees well with other recent reports (Comte et al., 2006; d’Abzac et al., 2010a). For the EPS using formaldehyde/ NaOH, the log KM values of both Peak T1 and Peak T2 were slightly higher for the origin of aerobic versus anaerobic sludge whereas the values of Peak C exhibited the opposite trend (i.e., anaerobic > aerobic). This result further suggests the existence of the structural heterogeneity with respect to the strength of Hg(II) binding for the EPS with different sludge formation conditions. 5. Conclusions More extraction rates of EPS were achieved by the formaldehyde/NaOH method while the EPS using CER showed characteristics associated with more aromatic and more condensed structures with higher molecular weight. The differences between the two extraction methods were more pronounced when the EPS of anaerobic sludge were compared with each other. The differences between the extraction methods and between the sludge formation conditions were possibly distinguished by comparing the EEM patterns (e.g., higher peak ratios of either T1/C or T2/C for the EPS using CER, and the exclusive presence of Peak A for the EPS of anaerobic sludge). The FT-IR spectra revealed that the formaldehyde/NaOH method could effectively extract the EPS components containing higher content of protein relative to carbohydrate as well as a higher abundance of acidic functional groups. The highest extent of pyrene binding was observed for the EPS of anaerobic sludge using CER, consistent with the implications from the other characteristics investigated. Stronger Hg (II) binding of the EPS using formaldehyde/NaOH versus CER appears to be related to the presence of more abundance of acidic functional groups. Relative differences in the stability constants calculated at each EEM peak were not consistent for the EPS with different sludge formation conditions, implying the existence of the EPS structural heterogeneity with respect to Hg(II) binding strength. Our results suggest that the choice of the extraction methods and the sludge formation conditions should be considered as critical factors for determining the EPS characteristics including their binding properties. Acknowledgments This work was supported by a National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-20110029028). Additional support was provided by Hi Seoul Science/ Humanities Fellowship from the Seoul Scholarship Foundation.

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