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© IWA Publishing 2015 Water Science & Technology
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Optimization for extracellular polymeric substances extraction of microbial aggregates Liang Zhu, Haitian Yu, Yimei Liu, Hanying Qi and Xiangyang Xu
ABSTRACT The extracellular polymeric substances (EPS) are important macromolecular components in microbial aggregates. The three EPS extraction methods – ultrasound þ cation exchange resins (CER) þ sulfide, ultrasound þ formamide þ NaOH, and ultrasound þ heat – were investigated in the study, and the component differences of extracted EPS from the loose flocs and dense aerobic granules were compared using chemical analysis and three-dimensional excitation-emission matrix (3D-EEM). Results showed that the contents of EPS were extracted effectively by ultrasound þ formamide þ NaOH and ultrasound þ heat methods, and the ultrasound þ CER þ sulfide method did not extract the polysaccharides (PS) or protein (PN) contents from the sludge samples. The 3D-EEM analysis indicated that the nature of peak B/D, peak C/E/F, and peak A/G were attributed to PN-like, humic acid-like and fulvic acid-like fluorophores. All fluorophores can be detected from the EPS extracted through the ultrasound þ heat method. Hopefully this will provide more information about the EPS interaction mechanism of microbial aggregates. Key words
| extracellular polymeric substances (EPS), extraction method, microbial aggregate, three-dimensional fluorescence spectrum technology
Liang Zhu (corresponding author) Haitian Yu Hanying Qi Xiangyang Xu Department of Environmental Engineering, Zhejiang University, Hangzhou, 310058, China E-mail: [email protected] Yimei Liu Hangzhou Environmental Monitoring Center, Hangzhou, 310007, China Liang Zhu Xiangyang Xu Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou, 310058, China
INTRODUCTION Extracellular polymeric substances (EPS) are important macromolecular components of microbial aggregates such as sludge flocs, biofilm, and granular sludge. Multi-species communities of micro-organisms could be embedded in the EPS matrix for self-protection from environmental stresses and improvement of settling performance (Flemming & Wingender ; Liu et al. ; McSwain et al. ; Sheng et al. ). In general, proteins (PNs) and polysaccharides (PS) are the major components of EPS in microbial aggregates, accounting for approximately 70–80% of the total EPS amount (McSwain et al. ), while nucleic acids, humic acids, lectins, lipids, and other polymers are present in lower levels (Dignac et al. ; D’Abzac et al. ). In addition, the sludge EPS could also provide the nutrients and energy for microbes in wastewater treatment systems (Lochmatter et al. ). There have been many methods reported for EPS extraction, such as physical methods (centrifugation, ultrasound, heat, etc.) (Goodwin & Forster ; Morgan & Forster ; Fang & Jia ) and chemical methods (ethylene diamine tetraacetic acid (EDTA), sulfide, alkaline, cation doi: 10.2166/wst.2015.043
exchange resins (CER), etc.) (Brown & Lester ; Nielsen & Keiding ; Sheng & Yu ). In recent years, a combination of methods has greatly improved the EPS extraction efficiency from sludge samples. Park & Novak () used three extraction methods for EPS extraction. The CER method was highly selective for Ca2þ- and Mg2þbound EPS and the base extraction method was efficient for Al-bound EPS, and the amino-acid composition of extracted EPS differed for the three methods. Adav & Lee () found that the ultrasound method followed by formamide and NaOH had a higher extraction efficiency than that of the alkaline extraction method for the EPS of aerobic granules. However, the amount and composition of EPS extracted from various microbial aggregates strongly depended on the extraction methods (Wingender et al. ; Alasonati & Slaveykova ; Lee et al. ). Three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy as a rapid, selective, and sensitive technique has been successfully applied in characterizing dissolved organic matter in water, soil, and sludge, such as tryptophan, tyrosin, humic, fulvic, microbial
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by-product-like compounds (Coble et al. ; Coble ; Her et al. ). It has been proven to be a useful technique for detecting the PN and humic substances of sludge EPS (Wingender et al. ). In this study, the EPS in two kinds of sludge was extracted by three combined methods, including ultrasound followed by formamide and NaOH, ultrasound followed by CER and sulfide, and ultrasound followed by heat treatment. The objective was to compare the amount and composition of extracted EPS from the loose flocs and dense aerobic granular sludge, using chemical analysis and 3D-EEM technique. It is hopeful to establish an efficient and comprehensive EPS extraction method and reveal the EPS mechanism in the formation of microbial aggregates.
MATERIALS AND METHODS Sludge samples Two kinds of sludge samples were chosen from a laboratoryscale sequencing batch reactor (SBR) and aerobic granular sludge reactor. The synthetic municipal wastewater of these two reactors was as follows: chemical oxygen 1 demand (COD) of 250 mg L1, NHþ 4 -N of 40 mg L , and 1 3 PO4 -P of 6 mg L . The sludge flocs were collected from a laboratory-scale SBR reactor with a volume of 3 L. The 3 average removal efficiencies of COD, NHþ 4 -N, and PO4 -P were 98.97 ± 2.19, 92.97 ± 3.14, and 87.27 ± 11.83%, respectively. The biomass concentration was 3.83–4.72 g mixed liquor suspended solids (MLSS) L1 and sludge
Figure 1
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Procedure of different EPS extraction protocols.
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volume index (SVI) was 170 mL g1. Flocs were loose in structure, and light in color. Aerobic granular sludge was taken from the middle of a laboratory-scale aerobic granular sludge reactor with a working volume of 6 L. The influent loading rates of COD, NHþ 4 -N, total nitrogen, and total phosphorus were 2.43–2.79, 0.15–0.23, 0.24–0.33, and 0.056–0.076 kg m3 d1, and the average removal efficiencies were 87.7–100, 84.6–95.3, 80.1–88.1, and 74.3–83.0%, respectively. The MLSS was 10.75–11.32 g L1 and the SVI was below 20 mL g1. Granules had a dense structure and a mean diameter of 2 mm. EPS extraction methods The sludge flocs and granular sludge were washed three times with deionized water, and then the granular sludge was cracked by a vortex mixer. All the sludge samples were pretreated with 10 cycles of ultrasound for 10 s at 40 W and 21 kHz, and an interval of 10 s in an ice bath, in order to homogenize sludge. The procedures of three EPS extraction methods are described in Figure 1. CER followed by sulfide procedure extraction method was developed by Park & Novak (). The dosage of CER (strongly acidic styrene type001 × 7, Naþ form, Dowex-50, pretreated with 0.1 M NaOH for a pH of 7.0) was approximately adjusted to 60 g CER gVSS1 (VSS ¼ volatile suspended solids). After CER extraction process was completed, the CER were separated from the solution via settling, and then the solution was resuspended in the sulfide solution (15 mM Na2S·9H2O, pH 7.5) under air-free condition by inserting N2. The
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whole extraction operation was carried out in a shaker under the conditions of 15 C and 250 rpm for 14 hours. Heat extraction was slightly modified from the method by Morgan et al. (). The sludge samples after ultrasound were pretreated in 80 C water bath for 45 minutes. Formamide combined with NaOH extraction was the same as the procedure of Adav & Lee (). Thirty-seven percent of formamide was added into the sludge sample after ultrasound pretreatment at 4 C for 1 hour, and then EPS was extracted by using 1 M NaOH (v/v ¼ 1:1) at 4 C for 3 hours. After each EPS extraction, the EPS was separated from treated sludge by centrifugation at 20,000 g and 4 C for 20 minutes, and the supernatant was collected as EPS extracts. All the samples were tested in triplicate.
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RESULTS AND DISCUSSION
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The amount and component of sludge EPS extracted by different methods
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Chemical analysis
The contents of PS and PN extracted from flocs and granular sludge by three extraction methods are summarized in Figure 2. Results show that the contents of PN extracted from flocs and granular sludge were as follows: ultrasound þ formamide þ NaOH method (I) > ultrasound þ heat method (II) > ultrasound þ CER þ sulfide method (III). The PN content extracted from flocs was four times that from granular sludge, using method III. The efficiencies of PS extracted by the three methods were similar to the PN content, and the PS extracted by method III was far lower than the other two methods. Nucleic acid content of EPS extracted from sludge was an indicator of cell lysis, and the results are
The dry weight of flocs and aerobic granular sludge was measured according to Standard Methods (APHA ). The carbohydrate content in the sludge EPS was measured by the phenol-sulfuric acid method with glucose as the standard (Dubois et al. ). The PN content in the sludge EPS was measured by the modified Lowry method using bovine serum albumin as the standard (Frolund et al. ). The nucleic acid content was taken as the indicator of cell lysis (Brown & Lester ), and was measured by the diphenylamine colorimetric method using fish DNA as the standard (Sun et al. ). 3D-EEM fluorescence spectroscopy The EEM spectra of sludge EPS was measured using the luminescence spectrometer (LS-55, Perkin-Elmer Co., USA) and collected with subsequent scanning emission spectra from 300 to 550 nm at 0.5 nm increments by varying the excitation wavelength (Ex) from 200 to 400 nm at 10 nm increments. Excitation and emission slits were maintained at 5 nm and the scanning speed was set at 1,200 nm min1 for all the measurements. A 290 nm emission cutoff filter was used to eliminate second-order Raleigh light scattering. The spectrum of double distilled water was recorded as the blank. The Origin 8.0 software was employed for analyzing the EEM data. The x-axis represented the emission spectra from 300 to 550 nm, and the y-axis was the Ex wavelength from 200 to 400 nm. Twenty contour lines as the third dimension were shown for each EEM spectrum to represent the fluorescence intensity (Sheng & Yu ).
Figure 2
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PN and PS contents of sludge EPS extracted by three methods. (a) PN; (b) PS. The error bars refer to the triplicate samples.
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presented in Figure 3. The quantities of DNA were all less than 1.5 mg gVSS1 for all the samples, which indicated negligible contamination of intracellular substances in
Figure 3
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DNA content of sludge EPS extracted by three methods. The error bars refer to the triplicate samples.
Figure 4
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EEM fluorescence spectra of EPS extracted from flocs.
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the EPS (Adav & Lee ; Alasonati & Slaveykova ). It was speculated that the Fe-bound PN content of sludge flocs was more than that of granular sludge. Park & Novak () found that most of Ca2þ and Mg2þ were removed while Fe and Al remained intact via CER extraction, suggesting that this method is highly selective for Ca2þ- and Mg2þ-bound EPS. In this study, this method was optimal because we added Ca2þ and Mg2þ in influent water during the process. The correlation between Fe in sludge and sulfide-extracted EPS indicated the selectivity of this method for Fe-bound EPS. CER and sulfide procedure could extract EPS linked with divalent cations and Fe, and sulfide extraction following CER increased the contact area and favored the extraction of Fe-bound PN. However, Li et al. () found that iron was neither temporally accumulated nor heterogeneously spatially distributed in aerobic granular sludge, and divalent cations could accumulate which result in lower content of Febound PN in granular sludge. The sludge flocs had a greater
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specific surface area which increases the contact area between iron and sludge (Adav & Lee ; D’Abzac et al. ). In the field of PS, the less negative charge of PS resulted in the weakness of binding characteristics between the PS and metal ion (D’Abzac et al. ; Zhu et al. ). According to the PS and PN contents obtained via the three methods, PN content extracted from flocs was four times of that using method III, and the PS extracted by method III was far lower than the other two methods. This was consistent with previous studies. Fluorescence spectra analysis of sludge EPS extracted by different methods The fluorescence spectra analysis of sludge EPS extracted by three methods from flocs and granular sludge are shown in Figures 4 and 5, respectively. Each 3D-EEM fluorescence spectrum demonstrated information about the aromatic PNlike (peak B, Ex/emission wavelength (Em) ¼ 230.0/350.0– 371.0 nm; peak D, Ex/Em ¼ 280.0–290.0/359.0–378.0 nm),
Figure 5
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EEM fluorescence spectra of EPS extracted from granular sludge.
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humic acid-like (peak C, Ex/Em ¼ 255.0/409.5 nm; peak E, Ex/Em ¼ 320.0/389.5–394.0 nm; peak F, Ex/Em ¼ 360.0– 365.0/446.0–455.0 nm), and fulvic acid-like (peak A, Ex/Em ¼ 230.0/386.0–394.0 nm; peak G, Ex/Em ¼ 240.0/ 444.5–449.5 nm) substances of the sludge EPS based on the 3D-EEM regionalism developed by Chen et al. (). Table 1 lists the excitation and emission parameters from each EEM fluorescence spectrum. The locations of peak G and peak F shifted toward lower wavelength to different extents on the emission and excitation scales of peak A and peak E, respectively. As reported previously (Chen et al. ), the humic acid-like and fulvic acid-like substances contained many carboxyl groups which react with NaOH, and result in a significant reduction of carboxyl groups. A blue shift was related to the elimination of particular functional groups such as carboxyl, amino, carbonyl, alkoxyl (Swietlik et al. ). Peak B and D of aromatic PN-like substances were not present in the 3D-EEM spectra of the sludge EPS from either flocs or granular sludge extracted by method I. Sheng & Yu
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Table 1
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Fluorescence spectra parameters of sludge EPS extracted by the different methods Sludge flocs
Aerobic granular sludge
Items
Peaks
Ex/Em (nm)
Peaks
Ex/Em (nm)
CERþsulfide
B D F G
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 240.0/444.5–449.5
B D F C
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 255.0/409.5
FormamideþNaOH
A E
230.0/386.0–394.0 320.0/389.5–394.0
A E
230.0/386.0–394.0 320.0/389.5–394.0
Heat
B D F G
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 240.0/444.5–449.5
B D F G
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 240.0/444.5–449.5
() found that both pH and EPS concentration were significant for peak location and intensity of EPS’s EEM fluorescence spectra, but did not bring about the peak disappearance. Wei et al. () investigated the correlation between fractional, biodegradable and spectral characteristics of sludge EPS by different protocols. Different extraction protocols preferably extracted EPS with distinct fractional, biodegradable, and spectral characteristics which could be applied in specific usages. It was suggested that method I could not efficiently extract the aromatic PN-like substance of EPS from either flocs or granular sludge. Fulvic acid-like substance was not present in the EPS samples extracted by method III from granular sludge, which indicated that method III is not very efficient for the extraction of fulvic acid-like substance. In contrast, the 3D-EEM spectra of sludge EPS extracted by the heat method included fluorescence peaks of all substances such as aromatic PN-like, fulvic acid-like, and humic acid-like. Although the heat extraction method may produce more cell fractionation, it does not affect the existence of substances. Results demonstrated that method II is better for EPS extraction from different sludge samples, and is helpful for further analysis of EPS function mechanism in the formation of microbial aggregates such as flocs, biofilms, and granular sludge (Frolund et al. ).
CONCLUSIONS The results of EPS amount and components showed that the ultrasound with formamide þ NaOH or heat methods were both efficient for sludge EPS extraction. Analysis of 3DEEM indicated that all peaks of EEM fluorescence spectra were attributed to PN-like (the nature of peak B and D),
humic acid-like (the nature of peak C, E, and F), and fulvic acid-like (the nature of peak A and G) fluorophores. Only the 3D-EEM of EPS from both sludge extracted by ultrasound þ heat method contained all the above fluorophores, and was the efficient EPS extraction method for sludge samples with different structures.
ACKNOWLEDGEMENTS This work was funded by the National Natural Science Foundation of China (Nos 51478416 and 51008269), Natural Science Foundation of Zhejiang province (No. LY13E080003), Public project in Zhejiang province (No. 2014C33017) and Fundamental Research Funds for the Central Universities.
REFERENCES Adav, S. S. & Lee, D. J. Extraction of extracellular polymeric substances from aerobic granule with compact interior structure. J. Hazard. Mater. 154 (1–3), 1120–1126. Alasonati, E. & Slaveykova, V. I. Effects of extraction methods on the composition and molar mass distributions of exopolymeric substances of the bacterium Sinorhizobium meliloti. Bioresour. Technol. 114, 603–609. APHA. Standard Methods for the Examination of Water and Wastewater, 20th edn. American Public Health Association, Washington, DC, USA. Brown, M. J. & Lester, J. N. Comparison of bacterial extracellular polymer extraction methods. Appl. Environ. Microbiol. 40 (2), 179–185. Chen, W., Westerhoff, P., Leenheer, J. A. & Booksh, K. Fluorescence excitation – emission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37 (24), 5701–5710.
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Coble, P. G. Characterization of marine and terrestrial DOM in seawater using excitation emission matrix spectroscopy. Mar. Chem. 51 (4), 325–346. Coble, P. G., Green, S. A., Blough, N. V. & Gagosian, R. B. Characterization of dissolved organic matter in the Black Sea by fluorescence spectroscopy. Nature 348 (6300), 432–435. D’Abzac, P. F., Bordas, F., van Hullebusch, E., Lens, P. N. L. & Guibaud, G. Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols. Appl. Microbiol. Biotechnol. 85 (5), 1589–1599. Dignac, M. F., Urbain, V., Rybacki, D., Bruchet, A., Snidaro, D. & Scribe, P. Chemical description of extracellular polymers: implication on activated sludge floc structure. Water Sci. Technol. 38 (8–9), 45–53. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28 (3), 350–356. Fang, H. P. & Jia, X. S. Extraction of extracellular polymer from anaerobic sludges. Biotechnol Technol. 10 (11), 803–808. Flemming, H. C. & Wingender, J. The biofilm matrix. Nat. Rev. Microbiol. 8 (9), 623–633. Frolund, B., Palmgren, R., Keiding, K. & Nielsen, P. H. Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res. 30 (8), 1749–1758. Goodwin, J. A. S. & Forster, C. F. A further examination into the composition of activated-sludge surfaces in relation to their settlement characteristics. Water Res. 19 (4), 527–533. Her, N., Amy, G., McKnight, D., Sohn, J. & Yoon, Y. Characterization of DOM as a function of MW by fluorescence EEM and HPLC-SEC using UVA, DOC, and fluorescence detection. Water Res. 37 (17), 4295–4303. Lee, B. M., Shin, H. S. & Hur, J. Comparison of the characteristics of extracellular polymeric substances for two different extraction methods and sludge formation conditions. Chemosphere 90, 237–244. Li, Z. H., Wang, X. C., Kuba, T. & Kusuda, T. Porous structure and spatial characteristics of aerobic granules. Int. J. Environ. Pollut. 45 (1–3), 25–35. Liu, Y. Q., Liu, Y. & Tay, J. H. The effects of extracellular polymeric substances on the formation and stability of biogranules. Appl. Microbiol. Biotechnol. 65, 143–148.
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Lochmatter, S., Gonzalez-Gil, G. & Holliger, C. Optimized aeration strategies for nitrogen and phosphorus removal with aerobic granular sludge. Water Res. 47, 6187–6197. McSwain, B. S., Irvine, R. L., Hausner, M. & Wilderer, P. A. Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge. Appl. Environ. Microbiol. 71 (2), 1051–1057. Morgan, J. W. & Forster, C. F. A comparative study of the sonication of anaerobic and activated sludges. J. Chem. Technol. Biotechnol. 55 (1), 53–58. Morgan, J. W., Forster, C. F. & Evison, L. A comparative study of the nature of biopolymers extracted from anaerobic and activated sludges. Water Res. 24 (6), 743–750. Nielsen, P. H. & Keiding, K. Disintegration of activated sludge flocs in presence of sulfide. Water Res. 32 (2), 313–320. Park, C. & Novak, J. T. Characterization of activated sludge exocellular polymers using several cation-associated extraction methods. Water Res. 41 (8), 1679–1688. Sheng, G. P. & Yu, H. Q. Characterization of extracellular polymeric substances of aerobic and anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy. Water Res. 40 (6), 1233–1239. Sheng, G. P., Yu, H. Q. & Li, X. Y. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28 (6), 882–894. Sun, Y. D., Clinkenbeard, K. D., Clarke, C., Cudd, L., Highlander, S. K. & Dabo, S. M. Pasteurella haemolytica leukotoxin induced apoptosis of bovine lymphocytes involves DNA fragmentation. Vet. Microbiol. 65 (2), 153–166. Swietlik, J., Dabrowska, A., Raczyk-Stanisławiak, U. & Nawrocki, J. Reactivity of natural organic matter fractions with chlorine dioxide and ozone. Water Res. 38 (3), 547–558. Wei, L. L., Wang, K., Zhao, Q. L., Jiang, J. Q., Kong, X. J. & Lee, D. J. Fractional, biodegradable and spectral characteristics of extracted and fractionated sludge extracellular polymeric substances. Water Res. 46 (14), 4387–4396. Wingender, J., Neu, T. R. & Flemming, H. C. Microbial Extracellular Polymeric Substances: Characterization, Structures and Function. Springer-Verlag, Berlin Heidelberg. Zhu, L., Xu, X. Y., Lv, M. L., Yu, Y. W. & Qi, H. Y. Role and significance of extracellular polymeric substances on the property of aerobic granule. Bioresour. Technol. 107, 46–54.
First received 15 July 2014; accepted in revised form 19 January 2015. Available online 20 February 2015
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© IWA Publishing 2015 Water Science & Technology
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71.7
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2015
Optimization for extracellular polymeric substances extraction of microbial aggregates Liang Zhu, Haitian Yu, Yimei Liu, Hanying Qi and Xiangyang Xu
ABSTRACT The extracellular polymeric substances (EPS) are important macromolecular components in microbial aggregates. The three EPS extraction methods – ultrasound þ cation exchange resins (CER) þ sulfide, ultrasound þ formamide þ NaOH, and ultrasound þ heat – were investigated in the study, and the component differences of extracted EPS from the loose flocs and dense aerobic granules were compared using chemical analysis and three-dimensional excitation-emission matrix (3D-EEM). Results showed that the contents of EPS were extracted effectively by ultrasound þ formamide þ NaOH and ultrasound þ heat methods, and the ultrasound þ CER þ sulfide method did not extract the polysaccharides (PS) or protein (PN) contents from the sludge samples. The 3D-EEM analysis indicated that the nature of peak B/D, peak C/E/F, and peak A/G were attributed to PN-like, humic acid-like and fulvic acid-like fluorophores. All fluorophores can be detected from the EPS extracted through the ultrasound þ heat method. Hopefully this will provide more information about the EPS interaction mechanism of microbial aggregates. Key words
| extracellular polymeric substances (EPS), extraction method, microbial aggregate, three-dimensional fluorescence spectrum technology
Liang Zhu (corresponding author) Haitian Yu Hanying Qi Xiangyang Xu Department of Environmental Engineering, Zhejiang University, Hangzhou, 310058, China E-mail: [email protected] Yimei Liu Hangzhou Environmental Monitoring Center, Hangzhou, 310007, China Liang Zhu Xiangyang Xu Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou, 310058, China
INTRODUCTION Extracellular polymeric substances (EPS) are important macromolecular components of microbial aggregates such as sludge flocs, biofilm, and granular sludge. Multi-species communities of micro-organisms could be embedded in the EPS matrix for self-protection from environmental stresses and improvement of settling performance (Flemming & Wingender ; Liu et al. ; McSwain et al. ; Sheng et al. ). In general, proteins (PNs) and polysaccharides (PS) are the major components of EPS in microbial aggregates, accounting for approximately 70–80% of the total EPS amount (McSwain et al. ), while nucleic acids, humic acids, lectins, lipids, and other polymers are present in lower levels (Dignac et al. ; D’Abzac et al. ). In addition, the sludge EPS could also provide the nutrients and energy for microbes in wastewater treatment systems (Lochmatter et al. ). There have been many methods reported for EPS extraction, such as physical methods (centrifugation, ultrasound, heat, etc.) (Goodwin & Forster ; Morgan & Forster ; Fang & Jia ) and chemical methods (ethylene diamine tetraacetic acid (EDTA), sulfide, alkaline, cation doi: 10.2166/wst.2015.043
exchange resins (CER), etc.) (Brown & Lester ; Nielsen & Keiding ; Sheng & Yu ). In recent years, a combination of methods has greatly improved the EPS extraction efficiency from sludge samples. Park & Novak () used three extraction methods for EPS extraction. The CER method was highly selective for Ca2þ- and Mg2þbound EPS and the base extraction method was efficient for Al-bound EPS, and the amino-acid composition of extracted EPS differed for the three methods. Adav & Lee () found that the ultrasound method followed by formamide and NaOH had a higher extraction efficiency than that of the alkaline extraction method for the EPS of aerobic granules. However, the amount and composition of EPS extracted from various microbial aggregates strongly depended on the extraction methods (Wingender et al. ; Alasonati & Slaveykova ; Lee et al. ). Three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy as a rapid, selective, and sensitive technique has been successfully applied in characterizing dissolved organic matter in water, soil, and sludge, such as tryptophan, tyrosin, humic, fulvic, microbial
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by-product-like compounds (Coble et al. ; Coble ; Her et al. ). It has been proven to be a useful technique for detecting the PN and humic substances of sludge EPS (Wingender et al. ). In this study, the EPS in two kinds of sludge was extracted by three combined methods, including ultrasound followed by formamide and NaOH, ultrasound followed by CER and sulfide, and ultrasound followed by heat treatment. The objective was to compare the amount and composition of extracted EPS from the loose flocs and dense aerobic granular sludge, using chemical analysis and 3D-EEM technique. It is hopeful to establish an efficient and comprehensive EPS extraction method and reveal the EPS mechanism in the formation of microbial aggregates.
MATERIALS AND METHODS Sludge samples Two kinds of sludge samples were chosen from a laboratoryscale sequencing batch reactor (SBR) and aerobic granular sludge reactor. The synthetic municipal wastewater of these two reactors was as follows: chemical oxygen 1 demand (COD) of 250 mg L1, NHþ 4 -N of 40 mg L , and 1 3 PO4 -P of 6 mg L . The sludge flocs were collected from a laboratory-scale SBR reactor with a volume of 3 L. The 3 average removal efficiencies of COD, NHþ 4 -N, and PO4 -P were 98.97 ± 2.19, 92.97 ± 3.14, and 87.27 ± 11.83%, respectively. The biomass concentration was 3.83–4.72 g mixed liquor suspended solids (MLSS) L1 and sludge
Figure 1
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Procedure of different EPS extraction protocols.
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volume index (SVI) was 170 mL g1. Flocs were loose in structure, and light in color. Aerobic granular sludge was taken from the middle of a laboratory-scale aerobic granular sludge reactor with a working volume of 6 L. The influent loading rates of COD, NHþ 4 -N, total nitrogen, and total phosphorus were 2.43–2.79, 0.15–0.23, 0.24–0.33, and 0.056–0.076 kg m3 d1, and the average removal efficiencies were 87.7–100, 84.6–95.3, 80.1–88.1, and 74.3–83.0%, respectively. The MLSS was 10.75–11.32 g L1 and the SVI was below 20 mL g1. Granules had a dense structure and a mean diameter of 2 mm. EPS extraction methods The sludge flocs and granular sludge were washed three times with deionized water, and then the granular sludge was cracked by a vortex mixer. All the sludge samples were pretreated with 10 cycles of ultrasound for 10 s at 40 W and 21 kHz, and an interval of 10 s in an ice bath, in order to homogenize sludge. The procedures of three EPS extraction methods are described in Figure 1. CER followed by sulfide procedure extraction method was developed by Park & Novak (). The dosage of CER (strongly acidic styrene type001 × 7, Naþ form, Dowex-50, pretreated with 0.1 M NaOH for a pH of 7.0) was approximately adjusted to 60 g CER gVSS1 (VSS ¼ volatile suspended solids). After CER extraction process was completed, the CER were separated from the solution via settling, and then the solution was resuspended in the sulfide solution (15 mM Na2S·9H2O, pH 7.5) under air-free condition by inserting N2. The
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whole extraction operation was carried out in a shaker under the conditions of 15 C and 250 rpm for 14 hours. Heat extraction was slightly modified from the method by Morgan et al. (). The sludge samples after ultrasound were pretreated in 80 C water bath for 45 minutes. Formamide combined with NaOH extraction was the same as the procedure of Adav & Lee (). Thirty-seven percent of formamide was added into the sludge sample after ultrasound pretreatment at 4 C for 1 hour, and then EPS was extracted by using 1 M NaOH (v/v ¼ 1:1) at 4 C for 3 hours. After each EPS extraction, the EPS was separated from treated sludge by centrifugation at 20,000 g and 4 C for 20 minutes, and the supernatant was collected as EPS extracts. All the samples were tested in triplicate.
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RESULTS AND DISCUSSION
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The amount and component of sludge EPS extracted by different methods
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Chemical analysis
The contents of PS and PN extracted from flocs and granular sludge by three extraction methods are summarized in Figure 2. Results show that the contents of PN extracted from flocs and granular sludge were as follows: ultrasound þ formamide þ NaOH method (I) > ultrasound þ heat method (II) > ultrasound þ CER þ sulfide method (III). The PN content extracted from flocs was four times that from granular sludge, using method III. The efficiencies of PS extracted by the three methods were similar to the PN content, and the PS extracted by method III was far lower than the other two methods. Nucleic acid content of EPS extracted from sludge was an indicator of cell lysis, and the results are
The dry weight of flocs and aerobic granular sludge was measured according to Standard Methods (APHA ). The carbohydrate content in the sludge EPS was measured by the phenol-sulfuric acid method with glucose as the standard (Dubois et al. ). The PN content in the sludge EPS was measured by the modified Lowry method using bovine serum albumin as the standard (Frolund et al. ). The nucleic acid content was taken as the indicator of cell lysis (Brown & Lester ), and was measured by the diphenylamine colorimetric method using fish DNA as the standard (Sun et al. ). 3D-EEM fluorescence spectroscopy The EEM spectra of sludge EPS was measured using the luminescence spectrometer (LS-55, Perkin-Elmer Co., USA) and collected with subsequent scanning emission spectra from 300 to 550 nm at 0.5 nm increments by varying the excitation wavelength (Ex) from 200 to 400 nm at 10 nm increments. Excitation and emission slits were maintained at 5 nm and the scanning speed was set at 1,200 nm min1 for all the measurements. A 290 nm emission cutoff filter was used to eliminate second-order Raleigh light scattering. The spectrum of double distilled water was recorded as the blank. The Origin 8.0 software was employed for analyzing the EEM data. The x-axis represented the emission spectra from 300 to 550 nm, and the y-axis was the Ex wavelength from 200 to 400 nm. Twenty contour lines as the third dimension were shown for each EEM spectrum to represent the fluorescence intensity (Sheng & Yu ).
Figure 2
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PN and PS contents of sludge EPS extracted by three methods. (a) PN; (b) PS. The error bars refer to the triplicate samples.
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presented in Figure 3. The quantities of DNA were all less than 1.5 mg gVSS1 for all the samples, which indicated negligible contamination of intracellular substances in
Figure 3
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DNA content of sludge EPS extracted by three methods. The error bars refer to the triplicate samples.
Figure 4
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EEM fluorescence spectra of EPS extracted from flocs.
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the EPS (Adav & Lee ; Alasonati & Slaveykova ). It was speculated that the Fe-bound PN content of sludge flocs was more than that of granular sludge. Park & Novak () found that most of Ca2þ and Mg2þ were removed while Fe and Al remained intact via CER extraction, suggesting that this method is highly selective for Ca2þ- and Mg2þ-bound EPS. In this study, this method was optimal because we added Ca2þ and Mg2þ in influent water during the process. The correlation between Fe in sludge and sulfide-extracted EPS indicated the selectivity of this method for Fe-bound EPS. CER and sulfide procedure could extract EPS linked with divalent cations and Fe, and sulfide extraction following CER increased the contact area and favored the extraction of Fe-bound PN. However, Li et al. () found that iron was neither temporally accumulated nor heterogeneously spatially distributed in aerobic granular sludge, and divalent cations could accumulate which result in lower content of Febound PN in granular sludge. The sludge flocs had a greater
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specific surface area which increases the contact area between iron and sludge (Adav & Lee ; D’Abzac et al. ). In the field of PS, the less negative charge of PS resulted in the weakness of binding characteristics between the PS and metal ion (D’Abzac et al. ; Zhu et al. ). According to the PS and PN contents obtained via the three methods, PN content extracted from flocs was four times of that using method III, and the PS extracted by method III was far lower than the other two methods. This was consistent with previous studies. Fluorescence spectra analysis of sludge EPS extracted by different methods The fluorescence spectra analysis of sludge EPS extracted by three methods from flocs and granular sludge are shown in Figures 4 and 5, respectively. Each 3D-EEM fluorescence spectrum demonstrated information about the aromatic PNlike (peak B, Ex/emission wavelength (Em) ¼ 230.0/350.0– 371.0 nm; peak D, Ex/Em ¼ 280.0–290.0/359.0–378.0 nm),
Figure 5
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EEM fluorescence spectra of EPS extracted from granular sludge.
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humic acid-like (peak C, Ex/Em ¼ 255.0/409.5 nm; peak E, Ex/Em ¼ 320.0/389.5–394.0 nm; peak F, Ex/Em ¼ 360.0– 365.0/446.0–455.0 nm), and fulvic acid-like (peak A, Ex/Em ¼ 230.0/386.0–394.0 nm; peak G, Ex/Em ¼ 240.0/ 444.5–449.5 nm) substances of the sludge EPS based on the 3D-EEM regionalism developed by Chen et al. (). Table 1 lists the excitation and emission parameters from each EEM fluorescence spectrum. The locations of peak G and peak F shifted toward lower wavelength to different extents on the emission and excitation scales of peak A and peak E, respectively. As reported previously (Chen et al. ), the humic acid-like and fulvic acid-like substances contained many carboxyl groups which react with NaOH, and result in a significant reduction of carboxyl groups. A blue shift was related to the elimination of particular functional groups such as carboxyl, amino, carbonyl, alkoxyl (Swietlik et al. ). Peak B and D of aromatic PN-like substances were not present in the 3D-EEM spectra of the sludge EPS from either flocs or granular sludge extracted by method I. Sheng & Yu
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Fluorescence spectra parameters of sludge EPS extracted by the different methods Sludge flocs
Aerobic granular sludge
Items
Peaks
Ex/Em (nm)
Peaks
Ex/Em (nm)
CERþsulfide
B D F G
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 240.0/444.5–449.5
B D F C
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 255.0/409.5
FormamideþNaOH
A E
230.0/386.0–394.0 320.0/389.5–394.0
A E
230.0/386.0–394.0 320.0/389.5–394.0
Heat
B D F G
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 240.0/444.5–449.5
B D F G
230.0/350.0–371.0 280.0–290.0/359.0–378.0 360.0–365.0/446.0–455.0 240.0/444.5–449.5
() found that both pH and EPS concentration were significant for peak location and intensity of EPS’s EEM fluorescence spectra, but did not bring about the peak disappearance. Wei et al. () investigated the correlation between fractional, biodegradable and spectral characteristics of sludge EPS by different protocols. Different extraction protocols preferably extracted EPS with distinct fractional, biodegradable, and spectral characteristics which could be applied in specific usages. It was suggested that method I could not efficiently extract the aromatic PN-like substance of EPS from either flocs or granular sludge. Fulvic acid-like substance was not present in the EPS samples extracted by method III from granular sludge, which indicated that method III is not very efficient for the extraction of fulvic acid-like substance. In contrast, the 3D-EEM spectra of sludge EPS extracted by the heat method included fluorescence peaks of all substances such as aromatic PN-like, fulvic acid-like, and humic acid-like. Although the heat extraction method may produce more cell fractionation, it does not affect the existence of substances. Results demonstrated that method II is better for EPS extraction from different sludge samples, and is helpful for further analysis of EPS function mechanism in the formation of microbial aggregates such as flocs, biofilms, and granular sludge (Frolund et al. ).
CONCLUSIONS The results of EPS amount and components showed that the ultrasound with formamide þ NaOH or heat methods were both efficient for sludge EPS extraction. Analysis of 3DEEM indicated that all peaks of EEM fluorescence spectra were attributed to PN-like (the nature of peak B and D),
humic acid-like (the nature of peak C, E, and F), and fulvic acid-like (the nature of peak A and G) fluorophores. Only the 3D-EEM of EPS from both sludge extracted by ultrasound þ heat method contained all the above fluorophores, and was the efficient EPS extraction method for sludge samples with different structures.
ACKNOWLEDGEMENTS This work was funded by the National Natural Science Foundation of China (Nos 51478416 and 51008269), Natural Science Foundation of Zhejiang province (No. LY13E080003), Public project in Zhejiang province (No. 2014C33017) and Fundamental Research Funds for the Central Universities.
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First received 15 July 2014; accepted in revised form 19 January 2015. Available online 20 February 2015
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