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Degradation of extracellular polymeric substances (EPS) extracted from activated sludge by low-concentration ozonation
Chemosphere 147 (2016) 248e255
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Degradation of extracellular polymeric substances (EPS) extracted from activated sludge by low-concentration ozonation Lu Meng, Jinying Xi*, Marvin Yeung Environmental Simulation and Pollution Control State Key Joint Laboratory, School of Environment, Tsinghua University, 100084 Beijing, China
h i g h l i g h t s Degradation of EPS by ozonation was studied to understand activated sludge property. Ozone reacts with protein in EPS by attacking amino acid residue on polypeptide chain. The polysaccharides/protein molecular weights decreased significantly after ozonation.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2015 Received in revised form 6 December 2015 Accepted 17 December 2015 Available online xxx
Reaction mechanisms between ozone and extracellular polymeric substances (EPS) can be the key of understanding the improvements in microbial aggregates properties by low-concentration ozonation. In this study, EPS are extracted from activated sludge and treated continuously by ozone gas at 270 ± 41 ppm. The reaction between ozone and EPS was investigated by observation of EPS component concentrations, functional groups and molecular weight distributions using UVeVis spectrometry, excitation-emission matrix fluorescence spectroscopy (EEM), high performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GCeMS). In a 12-hour-ozonation experiment, significant ozone consumption was observed in the first 4 h and protein concentration in EPS solution was reduced by 30 ± 12%. However, the polysaccharides concentration only had a slightly decrease at the end of the ozonation process. UVeVis spectra and EEM spectra results suggest that ozone removed protein and fluorescent matters (SMP and tryptophan-like aromatic protein) rapidly by attacking specific amino acid residues on polypeptide chain. After ozonation, the molecular weight of polysaccharide and protein dropped by 4 orders of magnitude according to HPSEC results. TOC concentration of EPS solution was reduced by 13 ± 2% after ozonation. The loss in TOC could be explained by the observation of volatile organic compounds such as carboxylic acids, aldehydes and ketones in the offgas detected by GCeMS. The results in this study can provide a better understanding towards the mechanisms of improvements in activated sludge properties by ozonation. © 2015 Elsevier Ltd. All rights reserved.
Handling Editor: Xiangru Zhang Keywords: Extracellular polymeric substances (EPS) Ozone Protein Polysaccharide
1. Introduction In biological wastewater treatment processes, extracellular polymeric substances (EPS) are produced by the microorganisms in bioreactors. EPS are a complex mixture of high molecular weight polymers, consisting of protein, polysaccharides, humic acids, lipids, and nucleic acids (Frølund et al., 1996; Flemming and Wingender, 2001b). The accumulation of EPS occurs by a series of different mechanisms including excretion, secretion, cell lysis, and
* Corresponding author. E-mail address: [email protected] (J. Xi). http://dx.doi.org/10.1016/j.chemosphere.2015.12.060 0045-6535/© 2015 Elsevier Ltd. All rights reserved.
sorption (Nielsen et al., 1997; Flemming and Wingender, 2001a). EPS form as a layer around microbial aggregates to provide a threedimensional protective matrix, usually described as “house of cells” (Flemming and Wingender, 2001a,b). It's reported that the proportion of EPS varies between 50% and 80% (w/w) of total biofilms weight, indicating that EPS is a main component of biofilms (De Beer and Stoodley, 2006). Many attempts have been made to explore chemical compositions and physicochemical properties of EPS, especially that of sludge flocs, biofilms and granular sludge (Morgan et al., 1990; Flemming and Wingender, 2001a,b; Sheng and Yu, 2006; Adav and Lee, 2008; Ni et al., 2009; Zhu et al., 2015). In recent years, researchers paid much attention to EPS for their influences on the microbial aggregate properties in wastewater
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treatment process. EPS were proved to play an important role in sludge dewatering performance (Morgan et al., 1990; Neyens et al., 2004). Vogt et al. (2000) found that there were two types of extracellular water in biofilms: free water and water entrapped by EPS, suggesting that EPS play a crucial part in cell dewaterability. It has been widely reported that the increase in sludge EPS results in the lowed sludge dewaterability, based on measurements of specific resistance to filtration (SFR) (Pere et al., 1993), capillary suction time (CST) (Pere et al., 1993) and floc strength (Eriksson and Alm, 1991). The amount of protein in EPS also influences sludge dewaterability, sometimes positively (Higgins and Novak, 1997a,b) and sometimes negatively (Murthy and Novak, 1999; Houghton et al., 2001). However, the effects of hydrophilic polysaccharides in EPS are mostly negative (Murthy and Novak, 1999), for b-polysaccharides are considered to perform as framework in EPS matrix, forming a three-dimension structure with high hydroscopicity (Adav and Lee, 2008). As high molecular weight polymeric substances, EPS were also reported to be responsible for membrane fouling in membrane bioreactors (MBR) (Chang et al., 2001; Drews et al., 2006a). Researchers have attributed the scaling of MBR modules to looselybound EPS (Wang et al., 2009), and polysaccharides of EPS are the key factor for membrane fouling (Drews et al., 2006b). All results above suggest that changes in EPS concentration and composition may lead to changes in microbial aggregate properties and reactor performance. For decades, ozonation has been introduced to improve microbial aggregates' properties in biological wastewater treatment system (Lee et al., 2005; Huang and Wu, 2008; Garcia-Perez et al., 2013). Sludge ozonation for reducing excess sludge has been successfully applied in full-scale industrial and municipal wastewater treatment plants, and is considered to be one of the most effective techniques (Müller et al., 1998). It has been combined with biological wastewater treatment process to reduce sludge production, or adopted as a powerful approach to improve dewaterability of sludge (Yasui and Shibata, 1994; Ahn et al., 2002; Lee et al., 2005). The amount of excess sludge was reduced to nearly zero without decrease in effluent quality under ozone dosage of 0.05 g-O3 g-SS 1 (Yasui and Shibata, 1994; Lee et al., 2005), and ozone dosage at 0.5 g-O3 g-SS 1 resulted in sludge mass reduction by mineralization as well as volume reduction by improvement in dewaterability (Ahn et al., 2002). Similar practices have also been found in MBR. Huang and Wu (2008) observed that membrane filterability was improved by ozonation with a dosage less than 0.7 mg-O3 g-SS 1. Liu et al. (2011) found that pre-ozonation with dosage at 1.5 mg L 1 raw water resulted in a satisfying organic matter removal in subsequent MBR, and a much lower increase rate of trans-membrane pressure. The positive effects of ozonation were also observed in some biological systems for waste gas treatment. Xi et al. (2015) introduced 180e220 mg m 3 ozone to a gas-phase biofilter and found that the biomass accumulation rate was effectively reduced without a drop in removal efficiency. Maldonado-Diaz and Arriaga (2015) demonstrated that 90 ppb ozone diminished the biofilm thickness without affecting cell viability in a gas-phase biofilter treating formaldehyde. Above all, low dosage ozonation is proved to result in reduction in accumulated biomass and an improvement in microbial aggregate properties like sludge dewaterability. Considering the importance of EPS to microbial aggregate properties and the effects of ozonation, it is reasonable to get a hypothesis that ozone may change EPS characters and then attribute to the improvements in microbial aggregate properties. Ozone may change EPS characters either by direct reacting with EPS or by changing microbial community structure which affects EPS production. Moreover, it's probably that the two pathways both take
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effects. However, it will be difficult to study the two pathways at the same time because the ozonation of EPS and the production of EPS by microorganisms are both dynamic processes. In this study, EPS was extracted from activated sludge and the direct reaction between EPS and ozone was systematically investigated. The characteristics of EPS in terms of components concentration, functional groups and molecular weight (MW) distributions were observed by UVeVis spectrometry, excitationemission matrix fluorescence spectroscopy (EEM), high performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GCeMS). The results provide a better understanding on the improvements in activated sludge properties by ozonation. 2. Materials and methods 2.1. Activated sludge collection and EPS extraction The activated sludge used in this study was obtained from Xiaojiahe wastewater treatment plant in Beijing, China. An A2O process is adopted in the plant. The sludge was collected from the recycled line of sludge, with a total suspended solids (TSS) concentration of 3500e5000 mg L 1, 65e70% volatile suspended solids (VSS) and sludge settlement ratio (SV30) of 25e30%. The collected sludge was transported to laboratory within 2 h for further analysis and EPS extraction. The EPS were extracted using a cation exchange resin (CER) method (Frølund et al., 1996). The sludge sample was washed using a buffer consisting of 2 mM Na3PO4, 4 mM NaH2PO4, 9 mM NaCl and 1 mM KCl at pH 7 by centrifugation at 2000 rpm for 15 min at 4 C. The sludge pellets were re-suspended to their original volume using the buffer mentioned above. The solution was transferred to an extraction beaker, and the CER (sodium form 732, Sinopharm chemical reagent Co., Ltd, China) was added with a dosage of 60 g gVSS 1. The suspension was stirred for 6 h at 300 rpm and 4 C. Afterwards the CER/sludge suspension was centrifuged at 4000 rpm, 4 C for 10 min in order to remove the CER, and the supernatant was centrifuged twice at 12,000 rpm, 4 C for 10 min in order to remove the remaining flocs. The supernatant was then filtered through 0.45 mm acetate cellulose membranes and used as the EPS for ozonation and further analysis. The extracted EPS polysaccharides, protein and TOC concentration was 64 ± 8 mg L 1, 302 ± 30 mg L 1 and 158 ± 7 mg L 1, respectively, equivalent to 7.7 ± 0.9 mg gVSS 1, 36.4 ± 3.6 mg gVSS 1 and 19.8 ± 0.8 mg gVSS 1, respectively, which are in the reasonable range according to reported results in a review article reported by Liu and Fang (2003). 2.2. Ozonation experiment conditions 1 L of EPS solution was fed into a 2.5 L ozone reactor (Fig. 1). An air pump pumped ambient air into a UV lamp which generated ozone continuously during the reaction time and the generated ozone was introduced into the reactor. The inlet and outlet ozone concentration was measured by an ozone detector. By adjusting the flow rate of inlet gas, the concentration of inlet ozone was controlled. An aerator was used to ensure sufficient reaction between ozone and EPS. The total reaction time was 12 h. The outlet gas was absorbed by a Tenax™ tube consisting of solid sorbents (Tenax™ TA, mesh 60e80) and a stainless steel sorbet tube (PerkineElmer, USA; O.D.: 6.9 mm; I.D.: 4.9 mm; length: 88.9 mm) for GCeMS analysis, and the Tenax™ tube was replaced by a new one every 2 h. Every 1 h, 10 ml of liquor was taken out as samples for chemical, UVeVis spectrometry, EEM and HPSEC analysis. The experiment was repeated three times independently to ensure
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Fig. 1. Schematic diagram of the experiment system.
2.3. Analytical methods The SS and VSS content of the sludge were determined according to the Standard Methods (APHA, 1995). The polysaccharides contents of EPS were measured using the anthrone method (DuBois et al., 1956) with glucose as a standard, while the protein content was measured using the Lowry method (Lowry et al., 1951) with bovine serum albumin (BSA) as a standard. The total organic carbon (TOC) concentration of EPS was measured by a TOC analyzer (TOC-VCPH, Shimadzu, Japan). The UVeVis absorption spectra were performed using a UV spectrophotometer (UV-2401, Shimadzu, Japan). A luminescence spectrometer (F-7000, Hitachi, Japan) was used to measure the EEM spectra. In this study, EEM spectra were collected with subsequent scanning emission spectra from 240 nm to 600 nm at 1 nm increments by varying the excitation wavelength from 220 nm to 450 nm at 5 nm increments. Excitation and emission slits were both 5 nm and the scanning speed was 30,000 nm min 1. A 290 nm emission cutoff filter was used to eliminate second order Raleigh light scattering. The spectra of MilliQ water were used as the blank. The software Origin7.5 was employed for handling EEM data. EEM spectra are illustrated as the elliptical shape of contours. The MW distribution of EPS was measured by a HPSEC (LC-20 AD, Shimadzu, Japan) equipped with a PDA detector (SPD-M20A, Shimadzu, Japan) and an RID-10 detector (Shimadzu, Japan). A TSK column (G3000PWXL, TOSOH, Japan) was used and the column temperature was maintained at 40 C. The injection volume was 100 ml, and Milli-Q water was used as the mobile phase at a flow rate of 0.5 ml min 1. The column was calibrated using PEG as standards. Thermal desorption unit ATD 650(PerkineElmer Corp., USA) coupled with a GCMS-QP2010Plus gas chromatograph (Shimadzu, Japan) equipped with a DB-5MS 60 m 0.32 mm 0.5 mm column (Agilent Technologies, USA) was used for the identification of volatile organic compounds (VOCs) produced during the ozonation process. ATD 650 was coupled to GCeMS via a valve and a transfer line maintained at 210 C and 215 C, respectively. The conditions utilized for the thermal desorption system were as follows: desorption temperature, 250 C; trap heat temperature 280 C; trap cool temperature 25 C; desorption time, 10 min. Argon was used
as carrier gas with a flow rate of 1.5 ml min 1. The GC oven temperature was 40 C (hold for 2 min). A stepwise programmed linear temperature ramping was as flows: (1) 5 C min 1 to 120 C (hold for 2 min), and (2) 10 C min 1 further to 260 C (hold for 5 min). Ion source temperature was kept at 200 C.
3. Results and discussion 3.1. Ozone consumption The inlet and outlet ozone concentration was measured, and the results are shown in Fig. 2. During the ozonation reaction process, the inlet ozone concentration was 270 ± 41 ppm. The initial outlet ozone concentration was 43 ± 21 ppm and increased rapidly in 4 h. In this period, ozone was heavily consumed, suggesting that ozone reacted intensely with liquor. Afterwards, the concentration difference between inlet and outlet ozone maintained at 90 ± 15 ppm, suggesting that ozonation reaction reached equilibrium.
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Ozone concentration(ppm)
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inlet outlet difference between inlet and outlet
350 300 250 200 150 100 50 0 0
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4
6
8
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Time (h) Fig. 2. Time course of inlet and outlet ozone concentrations during EPS ozonation.
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3.2. Variation of EPS components during ozonation process During ozonation process, the polysaccharides, protein and TOC contents of EPS behaved distinctly (Fig. 3). Polysaccharides concentration remained constant in the first 6 h but decreased slightly in the following hours, with a final concentration at 60 ± 8 mg L 1. Protein, however, performed distinctly. It is clear to say that protein underwent a two-stage ozonation process. At the first stage, which counts from 0 h to 4 h, protein concentration declined sharply, and at the end of this stage, protein concentration was cut down by 30 ± 12%. At the second stage (4e12 h), protein concentration stayed at 213 ± 15 mg L 1, suggesting that reaction between protein and ozone reached equilibrium. As for TOC concentration, similar changes can be found. TOC concentration also underwent a twostage process. At the first stage (0 h-4 h), TOC concentration dropped from 158 ± 7 mg L 1 to 138 ± 7 mg L 1, with a removal efficiency of 13 ± 2%. Afterwards, TOC concentration stayed at 140 ± 8 mg L 1 from 4 h to 12 h. The variation of protein and TOC concentrations matched well with ozone consumption, which can be concluded as that ozone was heavily consumed in the first 4 h to cut down protein and TOC concentrations, while polysaccharides concentration remained unchanged. However, considering the principles of polysaccharides and protein concentration measurement, further discussion should be proposed. The anthrone method, which is used in this study to determine polysaccharides, detects polysaccharides by combining with hexose and hexuronic acid. That is to say, if polysaccharides are cut off by ozone on the carbon chain and form lower MW ones, then can still be determined as polysaccharides as long as hexose and hexuronic acid exist. It was reported that one of the reactions of ozone with polysaccharides is a direct glycosidic bond cleavage reaction by the insertion of ozone into the anomeric CeH bond (Pan et al., 1981). Fragmentation of the hydrotrioxide yields aldonic acidlactones. The conversion to the lactone leads to a shorter length of the polysaccharides chain. Thus, a preliminary explanation for why polysaccharides concentration changed little during the ozonation process is that a cut-off of polysaccharides chain occurred and ones with lower MW appeared.
As for protein, the Lowry method detects protein by combining with tryptophan and tyrosine residues. If tryptophan and tyrosine residues in protein react with ozone and are destroyed in chemical structure, then the protein will not be detected. According to Sharma and Graham (2010), for peptides and proteins, oxidation by ozone occurs at the tyrosine, tryptophan, histidine, cysteine and methionine residues. It is also reported that the aromatic amino acids such as phenylalanine and tryptophan, have high reactivity with ozone (Pryor et al., 1984). Therefore, it is likely that ozone was heavily consumed at the first 4 h to react with tyrosine, tryptophan, and phenylalanine residues in protein. 3.3. Changes in function groups and MW distribution during ozonation process 3.3.1. UVeVis absorption spectra of EPS As can be seen in Fig. 4, the UVeVis absorption spectra of initial EPS (0 h) had two peaks, one at 220 nm and the other at 252 nm. According to Havel (1996), the peak at 220 nm attributed to n/p* transition of the amide bond, and the other one at 252 nm, attributed to the aromatic amino acids, such as tryptophan, tyrosine and phenylalanine. When EPS were treated with ozone, the wavelength and intensity of peaks in UVeVis absorption spectra varied significantly by time. The intensity of the peaks at 220 nm remained similar, indicating that the attack of ozone on the amide bond in the polypeptide chain was not significant. On the other hand, during the first 4 h, the intensity of peaks at 252 nm gradually decreased with a shift to shorter wavelengths and disappeared at the end of 4 h. This is resulted from the degradation of aromatic amino acid residues (tryptophan, tyrosine and phenylalanine) in proteins. 3.3.2. EEM spectra of EPS The three-dimensional EEM fluorescence spectra of EPS reacted with ozone by different time are shown in Fig. 5. Three peaks can be identified from the EEM fluorescence spectra at 0 h. Peak A, with excitation/emission wavelength (Ex/Em) of 280/300 nm, was attributed to the soluble microbial by products (SMP). Peak B,
350 protein
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TOC polysaccharides
250 200 150 100 50 0 0
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4
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10
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Fig. 3. Time course of protein, polysaccharides, and TOC concentrations in EPS solution during the ozonation process.
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Fig. 4. UV/Vis absorption spectra of EPS during ozonation process.
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located at Ex/Em of 280/320 nm, was attributed to the soluble microbial by products (SMP). Peak C, around Ex/Em of 225/320 nm, was attributed to the tryptophan-like aromatic protein (Chen et al., 2003). When treated with ozone, the EEM fluorescence spectra changed significantly. From 0 h to 4 h, intensities of EEM spectra decreased sharply. Meanwhile, the locations and intensities of fluorescence peaks obviously varied. Peak B, which resulted from SMP, gradually disappeared at the end of 4 h. Intensities of peak C which resulted from tryptophan-like aromatic protein decreased a lot, resulting hardly recognition of peak C. At the rest of time (4 he12 h), the EEM fluorescence spectra remained similar.
Conclusions can therefore be made: ozone removes fluorescence matters in EPS in the first 4 h, especially SMP and tryptophan-like aromatic protein. 3.3.3. MW distributions results MW distributions of EPS during ozonation process were measured using HPSEC, as shown in Fig. 6. Based on the HPSEC chromatograms, changes of MW distributions of EPS during ozonation process can be identified. The solid line represents UV254 signal, whose detection is corresponding to protein in EPS. The dash line represents RID-10 signal, whose detection corresponds to polysaccharides in EPS. The HPSEC chromatograms suggest a shift
Fig. 5. EEM fluorescence spectra of EPS at ozonation time of: (a) 0 h; (b) 1 h; (c) 2 h; (d) 4 h; (e) 6 h; (f) 12 h.
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of EPS MW distribution during ozonation process both in intensity and retention time. The initial EPS had two peaks with a retention time of 11 and 14 min, for protein and polysaccharides, equaling to MW of 31,715 kDa and 2615 kDa, respectively. After reaction with ozone, the peaks became broader with a shift to longer retention time and smaller MW. Meanwhile, the intensities of UV254 and RID-10 detectors decreased. By the end of ozonation (Fig. 6 (f)), the peak of polysaccharides became 28 min, while the retention time of protein ranged from 16 to 27 min (168 Da and 388 kDa, respectively), and most of the EPS components were in this range. Comparing to the initial EPS (0 h), the MW of polysaccharides and protein was decreased by 4 orders of magnitude after ozonation. Conclusions can be made that EPS are likely to be partially
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ozonolysised, leading to a decrease in peak intensities and a transformation into lower MW compounds. 3.4. Gaseous products formed during ozonation process GCeMS is employed to detect the VOCs formed during ozonation process. Carboxylic acids, aldehydes and ketones are the main products formed during ozonation process (Fig. S1. and Table S1 in Supplementary Data). The result is in accordance with other studies between ozone and nature organic matters (NOM), humic substances, and protein (Le Lacheur and Glaze, 1996; KasprzykHordern, 2003; Swietlik et al., 2004). Katai and Schuerch (1966) have demonstrated that ozone attacks hydroxyl groups at C2, C3
Fig. 6. Molecular weight distribution of EPS at ozonation time of: (a) 0 h; (b) 1 h; (c) 2 h; (d) 4 h; (e) 6 h; (f) 12 h.
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or C6 positions in polysaccharides to produce carbonyl groups, which is recognized to transform into carboxyl groups at ozone exposure. A quantitative analysis seems difficult in this study, but the formation of carboxylic acids, aldehydes and ketones can be brought about to be a fine explanation for the decrease of TOC of EPS. It is reasonable to consider that ozone attacks EPS to form carboxylic acids, aldehydes and ketones, and most of them are noticeably volatile and very easily biodegraded. 3.5. Descriptive mechanisms for explaining improvements in activated sludge properties by ozonation Based on the results above, descriptive mechanisms are proposed to explain the improvements in activated sludge properties under ozone dosage (e.g. excess sludge reduction and improvement in dewaterability). When treated with ozone, concentration of EPS decreases, macromolecular contents break down with a better solubility, compounds with lower MW and carboxylic acids, aldehydes, ketones are produced which are ready to volatilize or be used as substrates by activated sludge, together leading to a reduction of excess sludge. Polysaccharides chains are cut off to form lower MW ones, leading to a collapse of framework of EPS three-dimensional matrix, and the trap of water is weakened, thus improving the dewaterability of sludge. Although EPS used in this study are extracted from activated sludge, the same mechanisms may also exist in biofilm systems. A further study is still needed to explore the conversion of biofilm EPS by ozonation. 4. Conclusion In this study, the reaction between ozone and EPS was systematically investigated in terms of component concentrations, functional groups and MW distributions using UVeVis spectrometry, EEM, HPSEC, and GCeMS. Based on the results, the following specific conclusions can be drawn: (1) Ozone removed protein concentration and fluorescence matters (SMP and tryptophan-like aromatic protein) rapidly by attacking specific amino acid residues (e.g. tryptophan, tyrosine and phenylalanine residues) on protein chain, with a removal efficiency of 30 ± 12%; (2) Polysaccharide concentration decreased slightly at the end of the ozonation process which indicates polysaccharides are relatively stable compared with protein; (3) The average molecular weights of polysaccharide and protein decreased by 4 orders of magnitude after ozonation; (4) TOC concentration decreased by 13 ± 2% during ozonation process, and part of the reduced TOC are considered to be transformed into volatile organic compounds such as carboxylic acids, aldehydes and ketones in the off-gas; (5) The results in this study revealed the characteristics of reaction between ozone and EPS, providing a better understanding towards the improvements in activated sludge properties after ozonation. Acknowledgment This research is supported by National Natural Science Foundation of China (Grant No. 51378286) and the State Environmental Protection Key Laboratory of Microorganism Application and Risk Control. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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Degradation of extracellular polymeric substances (EPS) extracted from activated sludge by low-concentration ozonation Lu Meng, Jinying Xi*, Marvin Yeung Environmental Simulation and Pollution Control State Key Joint Laboratory, School of Environment, Tsinghua University, 100084 Beijing, China
h i g h l i g h t s Degradation of EPS by ozonation was studied to understand activated sludge property. Ozone reacts with protein in EPS by attacking amino acid residue on polypeptide chain. The polysaccharides/protein molecular weights decreased significantly after ozonation.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2015 Received in revised form 6 December 2015 Accepted 17 December 2015 Available online xxx
Reaction mechanisms between ozone and extracellular polymeric substances (EPS) can be the key of understanding the improvements in microbial aggregates properties by low-concentration ozonation. In this study, EPS are extracted from activated sludge and treated continuously by ozone gas at 270 ± 41 ppm. The reaction between ozone and EPS was investigated by observation of EPS component concentrations, functional groups and molecular weight distributions using UVeVis spectrometry, excitation-emission matrix fluorescence spectroscopy (EEM), high performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GCeMS). In a 12-hour-ozonation experiment, significant ozone consumption was observed in the first 4 h and protein concentration in EPS solution was reduced by 30 ± 12%. However, the polysaccharides concentration only had a slightly decrease at the end of the ozonation process. UVeVis spectra and EEM spectra results suggest that ozone removed protein and fluorescent matters (SMP and tryptophan-like aromatic protein) rapidly by attacking specific amino acid residues on polypeptide chain. After ozonation, the molecular weight of polysaccharide and protein dropped by 4 orders of magnitude according to HPSEC results. TOC concentration of EPS solution was reduced by 13 ± 2% after ozonation. The loss in TOC could be explained by the observation of volatile organic compounds such as carboxylic acids, aldehydes and ketones in the offgas detected by GCeMS. The results in this study can provide a better understanding towards the mechanisms of improvements in activated sludge properties by ozonation. © 2015 Elsevier Ltd. All rights reserved.
Handling Editor: Xiangru Zhang Keywords: Extracellular polymeric substances (EPS) Ozone Protein Polysaccharide
1. Introduction In biological wastewater treatment processes, extracellular polymeric substances (EPS) are produced by the microorganisms in bioreactors. EPS are a complex mixture of high molecular weight polymers, consisting of protein, polysaccharides, humic acids, lipids, and nucleic acids (Frølund et al., 1996; Flemming and Wingender, 2001b). The accumulation of EPS occurs by a series of different mechanisms including excretion, secretion, cell lysis, and
* Corresponding author. E-mail address: [email protected] (J. Xi). http://dx.doi.org/10.1016/j.chemosphere.2015.12.060 0045-6535/© 2015 Elsevier Ltd. All rights reserved.
sorption (Nielsen et al., 1997; Flemming and Wingender, 2001a). EPS form as a layer around microbial aggregates to provide a threedimensional protective matrix, usually described as “house of cells” (Flemming and Wingender, 2001a,b). It's reported that the proportion of EPS varies between 50% and 80% (w/w) of total biofilms weight, indicating that EPS is a main component of biofilms (De Beer and Stoodley, 2006). Many attempts have been made to explore chemical compositions and physicochemical properties of EPS, especially that of sludge flocs, biofilms and granular sludge (Morgan et al., 1990; Flemming and Wingender, 2001a,b; Sheng and Yu, 2006; Adav and Lee, 2008; Ni et al., 2009; Zhu et al., 2015). In recent years, researchers paid much attention to EPS for their influences on the microbial aggregate properties in wastewater
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treatment process. EPS were proved to play an important role in sludge dewatering performance (Morgan et al., 1990; Neyens et al., 2004). Vogt et al. (2000) found that there were two types of extracellular water in biofilms: free water and water entrapped by EPS, suggesting that EPS play a crucial part in cell dewaterability. It has been widely reported that the increase in sludge EPS results in the lowed sludge dewaterability, based on measurements of specific resistance to filtration (SFR) (Pere et al., 1993), capillary suction time (CST) (Pere et al., 1993) and floc strength (Eriksson and Alm, 1991). The amount of protein in EPS also influences sludge dewaterability, sometimes positively (Higgins and Novak, 1997a,b) and sometimes negatively (Murthy and Novak, 1999; Houghton et al., 2001). However, the effects of hydrophilic polysaccharides in EPS are mostly negative (Murthy and Novak, 1999), for b-polysaccharides are considered to perform as framework in EPS matrix, forming a three-dimension structure with high hydroscopicity (Adav and Lee, 2008). As high molecular weight polymeric substances, EPS were also reported to be responsible for membrane fouling in membrane bioreactors (MBR) (Chang et al., 2001; Drews et al., 2006a). Researchers have attributed the scaling of MBR modules to looselybound EPS (Wang et al., 2009), and polysaccharides of EPS are the key factor for membrane fouling (Drews et al., 2006b). All results above suggest that changes in EPS concentration and composition may lead to changes in microbial aggregate properties and reactor performance. For decades, ozonation has been introduced to improve microbial aggregates' properties in biological wastewater treatment system (Lee et al., 2005; Huang and Wu, 2008; Garcia-Perez et al., 2013). Sludge ozonation for reducing excess sludge has been successfully applied in full-scale industrial and municipal wastewater treatment plants, and is considered to be one of the most effective techniques (Müller et al., 1998). It has been combined with biological wastewater treatment process to reduce sludge production, or adopted as a powerful approach to improve dewaterability of sludge (Yasui and Shibata, 1994; Ahn et al., 2002; Lee et al., 2005). The amount of excess sludge was reduced to nearly zero without decrease in effluent quality under ozone dosage of 0.05 g-O3 g-SS 1 (Yasui and Shibata, 1994; Lee et al., 2005), and ozone dosage at 0.5 g-O3 g-SS 1 resulted in sludge mass reduction by mineralization as well as volume reduction by improvement in dewaterability (Ahn et al., 2002). Similar practices have also been found in MBR. Huang and Wu (2008) observed that membrane filterability was improved by ozonation with a dosage less than 0.7 mg-O3 g-SS 1. Liu et al. (2011) found that pre-ozonation with dosage at 1.5 mg L 1 raw water resulted in a satisfying organic matter removal in subsequent MBR, and a much lower increase rate of trans-membrane pressure. The positive effects of ozonation were also observed in some biological systems for waste gas treatment. Xi et al. (2015) introduced 180e220 mg m 3 ozone to a gas-phase biofilter and found that the biomass accumulation rate was effectively reduced without a drop in removal efficiency. Maldonado-Diaz and Arriaga (2015) demonstrated that 90 ppb ozone diminished the biofilm thickness without affecting cell viability in a gas-phase biofilter treating formaldehyde. Above all, low dosage ozonation is proved to result in reduction in accumulated biomass and an improvement in microbial aggregate properties like sludge dewaterability. Considering the importance of EPS to microbial aggregate properties and the effects of ozonation, it is reasonable to get a hypothesis that ozone may change EPS characters and then attribute to the improvements in microbial aggregate properties. Ozone may change EPS characters either by direct reacting with EPS or by changing microbial community structure which affects EPS production. Moreover, it's probably that the two pathways both take
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effects. However, it will be difficult to study the two pathways at the same time because the ozonation of EPS and the production of EPS by microorganisms are both dynamic processes. In this study, EPS was extracted from activated sludge and the direct reaction between EPS and ozone was systematically investigated. The characteristics of EPS in terms of components concentration, functional groups and molecular weight (MW) distributions were observed by UVeVis spectrometry, excitationemission matrix fluorescence spectroscopy (EEM), high performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GCeMS). The results provide a better understanding on the improvements in activated sludge properties by ozonation. 2. Materials and methods 2.1. Activated sludge collection and EPS extraction The activated sludge used in this study was obtained from Xiaojiahe wastewater treatment plant in Beijing, China. An A2O process is adopted in the plant. The sludge was collected from the recycled line of sludge, with a total suspended solids (TSS) concentration of 3500e5000 mg L 1, 65e70% volatile suspended solids (VSS) and sludge settlement ratio (SV30) of 25e30%. The collected sludge was transported to laboratory within 2 h for further analysis and EPS extraction. The EPS were extracted using a cation exchange resin (CER) method (Frølund et al., 1996). The sludge sample was washed using a buffer consisting of 2 mM Na3PO4, 4 mM NaH2PO4, 9 mM NaCl and 1 mM KCl at pH 7 by centrifugation at 2000 rpm for 15 min at 4 C. The sludge pellets were re-suspended to their original volume using the buffer mentioned above. The solution was transferred to an extraction beaker, and the CER (sodium form 732, Sinopharm chemical reagent Co., Ltd, China) was added with a dosage of 60 g gVSS 1. The suspension was stirred for 6 h at 300 rpm and 4 C. Afterwards the CER/sludge suspension was centrifuged at 4000 rpm, 4 C for 10 min in order to remove the CER, and the supernatant was centrifuged twice at 12,000 rpm, 4 C for 10 min in order to remove the remaining flocs. The supernatant was then filtered through 0.45 mm acetate cellulose membranes and used as the EPS for ozonation and further analysis. The extracted EPS polysaccharides, protein and TOC concentration was 64 ± 8 mg L 1, 302 ± 30 mg L 1 and 158 ± 7 mg L 1, respectively, equivalent to 7.7 ± 0.9 mg gVSS 1, 36.4 ± 3.6 mg gVSS 1 and 19.8 ± 0.8 mg gVSS 1, respectively, which are in the reasonable range according to reported results in a review article reported by Liu and Fang (2003). 2.2. Ozonation experiment conditions 1 L of EPS solution was fed into a 2.5 L ozone reactor (Fig. 1). An air pump pumped ambient air into a UV lamp which generated ozone continuously during the reaction time and the generated ozone was introduced into the reactor. The inlet and outlet ozone concentration was measured by an ozone detector. By adjusting the flow rate of inlet gas, the concentration of inlet ozone was controlled. An aerator was used to ensure sufficient reaction between ozone and EPS. The total reaction time was 12 h. The outlet gas was absorbed by a Tenax™ tube consisting of solid sorbents (Tenax™ TA, mesh 60e80) and a stainless steel sorbet tube (PerkineElmer, USA; O.D.: 6.9 mm; I.D.: 4.9 mm; length: 88.9 mm) for GCeMS analysis, and the Tenax™ tube was replaced by a new one every 2 h. Every 1 h, 10 ml of liquor was taken out as samples for chemical, UVeVis spectrometry, EEM and HPSEC analysis. The experiment was repeated three times independently to ensure
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Fig. 1. Schematic diagram of the experiment system.
2.3. Analytical methods The SS and VSS content of the sludge were determined according to the Standard Methods (APHA, 1995). The polysaccharides contents of EPS were measured using the anthrone method (DuBois et al., 1956) with glucose as a standard, while the protein content was measured using the Lowry method (Lowry et al., 1951) with bovine serum albumin (BSA) as a standard. The total organic carbon (TOC) concentration of EPS was measured by a TOC analyzer (TOC-VCPH, Shimadzu, Japan). The UVeVis absorption spectra were performed using a UV spectrophotometer (UV-2401, Shimadzu, Japan). A luminescence spectrometer (F-7000, Hitachi, Japan) was used to measure the EEM spectra. In this study, EEM spectra were collected with subsequent scanning emission spectra from 240 nm to 600 nm at 1 nm increments by varying the excitation wavelength from 220 nm to 450 nm at 5 nm increments. Excitation and emission slits were both 5 nm and the scanning speed was 30,000 nm min 1. A 290 nm emission cutoff filter was used to eliminate second order Raleigh light scattering. The spectra of MilliQ water were used as the blank. The software Origin7.5 was employed for handling EEM data. EEM spectra are illustrated as the elliptical shape of contours. The MW distribution of EPS was measured by a HPSEC (LC-20 AD, Shimadzu, Japan) equipped with a PDA detector (SPD-M20A, Shimadzu, Japan) and an RID-10 detector (Shimadzu, Japan). A TSK column (G3000PWXL, TOSOH, Japan) was used and the column temperature was maintained at 40 C. The injection volume was 100 ml, and Milli-Q water was used as the mobile phase at a flow rate of 0.5 ml min 1. The column was calibrated using PEG as standards. Thermal desorption unit ATD 650(PerkineElmer Corp., USA) coupled with a GCMS-QP2010Plus gas chromatograph (Shimadzu, Japan) equipped with a DB-5MS 60 m 0.32 mm 0.5 mm column (Agilent Technologies, USA) was used for the identification of volatile organic compounds (VOCs) produced during the ozonation process. ATD 650 was coupled to GCeMS via a valve and a transfer line maintained at 210 C and 215 C, respectively. The conditions utilized for the thermal desorption system were as follows: desorption temperature, 250 C; trap heat temperature 280 C; trap cool temperature 25 C; desorption time, 10 min. Argon was used
as carrier gas with a flow rate of 1.5 ml min 1. The GC oven temperature was 40 C (hold for 2 min). A stepwise programmed linear temperature ramping was as flows: (1) 5 C min 1 to 120 C (hold for 2 min), and (2) 10 C min 1 further to 260 C (hold for 5 min). Ion source temperature was kept at 200 C.
3. Results and discussion 3.1. Ozone consumption The inlet and outlet ozone concentration was measured, and the results are shown in Fig. 2. During the ozonation reaction process, the inlet ozone concentration was 270 ± 41 ppm. The initial outlet ozone concentration was 43 ± 21 ppm and increased rapidly in 4 h. In this period, ozone was heavily consumed, suggesting that ozone reacted intensely with liquor. Afterwards, the concentration difference between inlet and outlet ozone maintained at 90 ± 15 ppm, suggesting that ozonation reaction reached equilibrium.
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Ozone concentration(ppm)
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inlet outlet difference between inlet and outlet
350 300 250 200 150 100 50 0 0
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4
6
8
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Time (h) Fig. 2. Time course of inlet and outlet ozone concentrations during EPS ozonation.
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3.2. Variation of EPS components during ozonation process During ozonation process, the polysaccharides, protein and TOC contents of EPS behaved distinctly (Fig. 3). Polysaccharides concentration remained constant in the first 6 h but decreased slightly in the following hours, with a final concentration at 60 ± 8 mg L 1. Protein, however, performed distinctly. It is clear to say that protein underwent a two-stage ozonation process. At the first stage, which counts from 0 h to 4 h, protein concentration declined sharply, and at the end of this stage, protein concentration was cut down by 30 ± 12%. At the second stage (4e12 h), protein concentration stayed at 213 ± 15 mg L 1, suggesting that reaction between protein and ozone reached equilibrium. As for TOC concentration, similar changes can be found. TOC concentration also underwent a twostage process. At the first stage (0 h-4 h), TOC concentration dropped from 158 ± 7 mg L 1 to 138 ± 7 mg L 1, with a removal efficiency of 13 ± 2%. Afterwards, TOC concentration stayed at 140 ± 8 mg L 1 from 4 h to 12 h. The variation of protein and TOC concentrations matched well with ozone consumption, which can be concluded as that ozone was heavily consumed in the first 4 h to cut down protein and TOC concentrations, while polysaccharides concentration remained unchanged. However, considering the principles of polysaccharides and protein concentration measurement, further discussion should be proposed. The anthrone method, which is used in this study to determine polysaccharides, detects polysaccharides by combining with hexose and hexuronic acid. That is to say, if polysaccharides are cut off by ozone on the carbon chain and form lower MW ones, then can still be determined as polysaccharides as long as hexose and hexuronic acid exist. It was reported that one of the reactions of ozone with polysaccharides is a direct glycosidic bond cleavage reaction by the insertion of ozone into the anomeric CeH bond (Pan et al., 1981). Fragmentation of the hydrotrioxide yields aldonic acidlactones. The conversion to the lactone leads to a shorter length of the polysaccharides chain. Thus, a preliminary explanation for why polysaccharides concentration changed little during the ozonation process is that a cut-off of polysaccharides chain occurred and ones with lower MW appeared.
As for protein, the Lowry method detects protein by combining with tryptophan and tyrosine residues. If tryptophan and tyrosine residues in protein react with ozone and are destroyed in chemical structure, then the protein will not be detected. According to Sharma and Graham (2010), for peptides and proteins, oxidation by ozone occurs at the tyrosine, tryptophan, histidine, cysteine and methionine residues. It is also reported that the aromatic amino acids such as phenylalanine and tryptophan, have high reactivity with ozone (Pryor et al., 1984). Therefore, it is likely that ozone was heavily consumed at the first 4 h to react with tyrosine, tryptophan, and phenylalanine residues in protein. 3.3. Changes in function groups and MW distribution during ozonation process 3.3.1. UVeVis absorption spectra of EPS As can be seen in Fig. 4, the UVeVis absorption spectra of initial EPS (0 h) had two peaks, one at 220 nm and the other at 252 nm. According to Havel (1996), the peak at 220 nm attributed to n/p* transition of the amide bond, and the other one at 252 nm, attributed to the aromatic amino acids, such as tryptophan, tyrosine and phenylalanine. When EPS were treated with ozone, the wavelength and intensity of peaks in UVeVis absorption spectra varied significantly by time. The intensity of the peaks at 220 nm remained similar, indicating that the attack of ozone on the amide bond in the polypeptide chain was not significant. On the other hand, during the first 4 h, the intensity of peaks at 252 nm gradually decreased with a shift to shorter wavelengths and disappeared at the end of 4 h. This is resulted from the degradation of aromatic amino acid residues (tryptophan, tyrosine and phenylalanine) in proteins. 3.3.2. EEM spectra of EPS The three-dimensional EEM fluorescence spectra of EPS reacted with ozone by different time are shown in Fig. 5. Three peaks can be identified from the EEM fluorescence spectra at 0 h. Peak A, with excitation/emission wavelength (Ex/Em) of 280/300 nm, was attributed to the soluble microbial by products (SMP). Peak B,
350 protein
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250 200 150 100 50 0 0
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4
6 Time (h)
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10
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Fig. 3. Time course of protein, polysaccharides, and TOC concentrations in EPS solution during the ozonation process.
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Fig. 4. UV/Vis absorption spectra of EPS during ozonation process.
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located at Ex/Em of 280/320 nm, was attributed to the soluble microbial by products (SMP). Peak C, around Ex/Em of 225/320 nm, was attributed to the tryptophan-like aromatic protein (Chen et al., 2003). When treated with ozone, the EEM fluorescence spectra changed significantly. From 0 h to 4 h, intensities of EEM spectra decreased sharply. Meanwhile, the locations and intensities of fluorescence peaks obviously varied. Peak B, which resulted from SMP, gradually disappeared at the end of 4 h. Intensities of peak C which resulted from tryptophan-like aromatic protein decreased a lot, resulting hardly recognition of peak C. At the rest of time (4 he12 h), the EEM fluorescence spectra remained similar.
Conclusions can therefore be made: ozone removes fluorescence matters in EPS in the first 4 h, especially SMP and tryptophan-like aromatic protein. 3.3.3. MW distributions results MW distributions of EPS during ozonation process were measured using HPSEC, as shown in Fig. 6. Based on the HPSEC chromatograms, changes of MW distributions of EPS during ozonation process can be identified. The solid line represents UV254 signal, whose detection is corresponding to protein in EPS. The dash line represents RID-10 signal, whose detection corresponds to polysaccharides in EPS. The HPSEC chromatograms suggest a shift
Fig. 5. EEM fluorescence spectra of EPS at ozonation time of: (a) 0 h; (b) 1 h; (c) 2 h; (d) 4 h; (e) 6 h; (f) 12 h.
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of EPS MW distribution during ozonation process both in intensity and retention time. The initial EPS had two peaks with a retention time of 11 and 14 min, for protein and polysaccharides, equaling to MW of 31,715 kDa and 2615 kDa, respectively. After reaction with ozone, the peaks became broader with a shift to longer retention time and smaller MW. Meanwhile, the intensities of UV254 and RID-10 detectors decreased. By the end of ozonation (Fig. 6 (f)), the peak of polysaccharides became 28 min, while the retention time of protein ranged from 16 to 27 min (168 Da and 388 kDa, respectively), and most of the EPS components were in this range. Comparing to the initial EPS (0 h), the MW of polysaccharides and protein was decreased by 4 orders of magnitude after ozonation. Conclusions can be made that EPS are likely to be partially
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ozonolysised, leading to a decrease in peak intensities and a transformation into lower MW compounds. 3.4. Gaseous products formed during ozonation process GCeMS is employed to detect the VOCs formed during ozonation process. Carboxylic acids, aldehydes and ketones are the main products formed during ozonation process (Fig. S1. and Table S1 in Supplementary Data). The result is in accordance with other studies between ozone and nature organic matters (NOM), humic substances, and protein (Le Lacheur and Glaze, 1996; KasprzykHordern, 2003; Swietlik et al., 2004). Katai and Schuerch (1966) have demonstrated that ozone attacks hydroxyl groups at C2, C3
Fig. 6. Molecular weight distribution of EPS at ozonation time of: (a) 0 h; (b) 1 h; (c) 2 h; (d) 4 h; (e) 6 h; (f) 12 h.
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or C6 positions in polysaccharides to produce carbonyl groups, which is recognized to transform into carboxyl groups at ozone exposure. A quantitative analysis seems difficult in this study, but the formation of carboxylic acids, aldehydes and ketones can be brought about to be a fine explanation for the decrease of TOC of EPS. It is reasonable to consider that ozone attacks EPS to form carboxylic acids, aldehydes and ketones, and most of them are noticeably volatile and very easily biodegraded. 3.5. Descriptive mechanisms for explaining improvements in activated sludge properties by ozonation Based on the results above, descriptive mechanisms are proposed to explain the improvements in activated sludge properties under ozone dosage (e.g. excess sludge reduction and improvement in dewaterability). When treated with ozone, concentration of EPS decreases, macromolecular contents break down with a better solubility, compounds with lower MW and carboxylic acids, aldehydes, ketones are produced which are ready to volatilize or be used as substrates by activated sludge, together leading to a reduction of excess sludge. Polysaccharides chains are cut off to form lower MW ones, leading to a collapse of framework of EPS three-dimensional matrix, and the trap of water is weakened, thus improving the dewaterability of sludge. Although EPS used in this study are extracted from activated sludge, the same mechanisms may also exist in biofilm systems. A further study is still needed to explore the conversion of biofilm EPS by ozonation. 4. Conclusion In this study, the reaction between ozone and EPS was systematically investigated in terms of component concentrations, functional groups and MW distributions using UVeVis spectrometry, EEM, HPSEC, and GCeMS. Based on the results, the following specific conclusions can be drawn: (1) Ozone removed protein concentration and fluorescence matters (SMP and tryptophan-like aromatic protein) rapidly by attacking specific amino acid residues (e.g. tryptophan, tyrosine and phenylalanine residues) on protein chain, with a removal efficiency of 30 ± 12%; (2) Polysaccharide concentration decreased slightly at the end of the ozonation process which indicates polysaccharides are relatively stable compared with protein; (3) The average molecular weights of polysaccharide and protein decreased by 4 orders of magnitude after ozonation; (4) TOC concentration decreased by 13 ± 2% during ozonation process, and part of the reduced TOC are considered to be transformed into volatile organic compounds such as carboxylic acids, aldehydes and ketones in the off-gas; (5) The results in this study revealed the characteristics of reaction between ozone and EPS, providing a better understanding towards the improvements in activated sludge properties after ozonation. Acknowledgment This research is supported by National Natural Science Foundation of China (Grant No. 51378286) and the State Environmental Protection Key Laboratory of Microorganism Application and Risk Control. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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