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Enhanced storage stability of aerobic granules seeded with pellets
Bioresource Technology 101 (2010) 8031–8037
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced storage stability of aerobic granules seeded with pellets Hua-Cheng Xu, Pin-Jing He *, Guan-Zhao Wang, Guang-Hui Yu, Li-Ming Shao State Key Laboratory of Pollution Control and Resource Reuse, Key Laboratory of Yangtze River Water Environment, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China
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Article history: Received 25 August 2009 Received in revised form 1 February 2010 Accepted 19 May 2010 Available online 17 June 2010 Keywords: Activated sludge flocs Aerobic granules Extracellular polymeric substances Pellets Storage stability
a b s t r a c t The responses of two different types of aerobic granules to storage, granule A seeded with activated sludge flocs and granule B seeded with pellets (cells), were investigated in this study. After 3-week storage, the surface of granule B remained compact and smooth while obvious crevices were observed on that of granule A. Compared with granule B, granule A had more decrease in biomass concentration, settleability, hydrophobicity, and extracellular polymeric substances (EPS) concentration after the storage. Results indicated that the stability loss of aerobic granules could be related to protein concentration decrease in the TB-EPS fraction and to protein framework disintegration in whole granule. Compared with aerobic granules seeded with activated sludge flocs, those seeded with pellets were more resistant against storage, and thus would have greater potential in practical applications. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Aerobic granulation is a novel self-immobilization process of microorganisms. Compared with conventional activated sludge, aerobic granules have the advantages of better settleability, stronger microbial structure, higher biomass retention, and better ability to handle toxic compounds (Adav et al., 2008a; Beun et al., 1999; Liu and Tay, 2002; Morgenroth et al., 1997). The application of aerobic granules was regarded as one of the promising biotechnologies in wastewater treatment (Adav et al., 2008b). Unfortunately, aerobic granules could easily lose stability and activity during storage, which would be the serious barrier to their practical application (Adav et al., 2008a; Zhang et al., 2005). Tay et al. (2002a) and Ng (2002) noted that the granules became more irregular and smaller and released soluble organic materials after storage for 8 weeks. Tay et al. (2002b) further showed that glucose-fed aerobic granules lost about 60% metabolic activity and acetate-fed granules lost about 90% metabolic activity after storing for 4 months at 4 °C in tap water. Lee et al. (2009) reported the endogenous respiration in the core of granules after starved for one month. Some storage methods were shown to improve the stability, e.g., Adav et al. (2007) proposed that storage at subfreezing temperatures ( 20 °C) was an ideal method for preserving granule stability and activity, furthermore, the addition of toxic substance (phenol) in the storing solution was beneficial to preserve the granule stability. However, this method is not quite practical be* Corresponding author. Tel./fax: +86 21 6598 6104. E-mail address: [email protected] (P.-J. He). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.05.062
cause the structure of granules would deteriorate by freezing and thawing. Although granule stability loss has been widely investigated, the information about the storage stability of aerobic granules seeded with different inocula is hardly found in the literatures so far. In most studies, aerobic granules were cultivated with activated sludge seed (Adav et al., 2008a). It is widely known that activated sludge flocs are mainly composed of cells and extracellular polymeric substances (EPS). Recently, Yu et al. (2008a, b) employed a novel EPS fractionation approach to obtain the pellets (cells) by extracting the EPS matrix. In this study, aerobic granules seeded with activated sludge flocs and pellets, respectively, were cultivated in two sequencing batch reactors (SBRs), then these two kinds of granules were both stored at 25 ± 1 °C for 3 weeks. The main purposes of this study were to investigate their responses to storage and explore the mechanism of stability loss. The EPS concentration variation during storage was monitored, and scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also applied to investigate the microstructure and distribution patterns of EPS and cells. Increased knowledge on this issue would further deepen our understanding of granule stability and be useful for the application of aerobic granules. 2. Methods 2.1. The cultivation and storage of aerobic granules Aerobic granules seeded with activated sludge flocs (granule A) and aerobic granules seeded with pellets (granule B) were
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cultivated in two SBRs, respectively. Each reactor had the same geometrical configuration with an internal diameter of 5 cm and the working H/D of 20. Fine air bubbles for aeration were introduced through porous stone-type diffuser (ACO-001, Sensen Co., Zhejiang, China) at the bottom of the column, giving a superficial gas velocity of 2.5 cm s 1. The two reactors were operated sequentially with a cycle time of 4 h (5 min of influent filling, 227 min of aeration, 3 min of settling and 5 min of effluent withdrawal). Effluent was discharged at the middle sampling port of the working column, and the hydraulic retention time (HRT) was 8 h. The reactors were housed in a temperature-controlled room at 25 ± 1 °C. The synthetic wastewater consisted of: 2000 mg/L sodium acetate; 1000 mg/L (NH4)2SO4, 200 mg/L MgCl2, 100 mg/L NaCl, 20 mg/L FeCl3, 10 mg/L CaCl2, 400 mg/L peptone, and other necessary micronutrients, and was similar to the synthetic wastewater used by Moy et al. (2002). Stable granules were both formed in the two reactors after operation for 25 days. The cultivated mature granules (named original granules) were then stored at room temperature (25 ± 1 °C) for 3 weeks with neither aeration nor nutrients. 2.2. Fractionation protocol for aerobic granules The fractionation procedures for sludge were carried out using protocols from Yu et al. (2008a, b). The flow chart of fractionation protocol is presented in Fig. 1. 2.3. Fluorescence staining and confocal laser scanning microscopy (CLSM) observation The collected original and stored granules were stained with fluorescent dyes in order to simultaneously visualize the distribution of proteins, a-polysaccharide, total cells, and dead cells in samples according to the scheme of Chen et al. (2007). The detailed approaches were: (1) Syto 63 (20 lM, 100 ll) was added to the samples, mixed on a shaker table (100 rpm) for 30 min and then washed twice with PBS solution. (2) NaHCO3 buffer (0.1 M, 100 ll) was added to maintain solution at pH 9, followed by adding a FITC solution (10 g/L, 10 ll), and the mixture was stirred for 1 h and washed twice with PBS solution. (3) The Con A solution (250 mg/L, 100 ll) was added to the samples for another 30 min and then washed twice with PBS solution. (4) The Sytox Blue solu-
tion (2.5 lM, 100 ll) was incubated with the samples for 10 min. All probes were purchased from Molecular Probes, Eugene, USA. The stained samples were frozen at 20 °C and immersed in Tissue-Tek 4583 O.C.T compound (Sakura). The 20 lm sections were then cut on a cryomicrotome (Cyrotome E, Thermo Shandon Limited, UK) for CLSM (Leica TCS SP2 confocal spectral microscope imaging system, Germany) observation. The samples were imaged with a 10 or 20 objective. 2.4. Scanning electron microscopy (SEM) observation Stored granules for SEM were: (1) Fixed with 2.5% glutaraldehyde for 2 h at room temperature. (2) Suspended in a 1% osmium tetroxide for 2 h. (3) Dehydrated via a graded series of ethanol solutions (50%, 70%, 90%, and 100%). (4) Subjected to critical point drying (Emitech, K850, UK) and coated with platinum by using a magnetron sputter coater (BEL-TEC, SCD 050 Sputter). (5) The granule structure and surface morphology were viewed via SEM (Quanta 200FEG). 2.5. Three-dimensional excitation and emission matrix (EEM) fluorescence spectroscopy The cuvettes were rinsed and ultrasonicated using 5% (w/w) nitric acid solution before analysis. All EEM spectra were measured with a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA, USA). EEM spectra were gathered with subsequent scanning emission spectra from 250 to 600 nm at 2 nm increments by varying the excitation wavelength from 200 to 500 nm at 10 nm increments. The spectra were recorded at a scan rate of 1200 nm/ min, using excitation and emission slit bandwidths of 5 nm. The voltage of the photomultiplier tube (PMT) was set to 800 V for high level light detection. Second-order Rayleigh and Raman scattering were filtered out (Lu et al., 2009). The blank scans were performed at intervals of 10 analyses using Milli-Q water. Specific fluorescence intensity (SFI) was obtained by dividing fluorescence intensity by dissolved organic carbon (DOC) concentration and reported in arbitrary units/(mg/L) DOC (AU/(mg/L) DOC). 2.6. Other analytical techniques Cell hydrophobicity was determined by the method described by Rosenberg et al. (1980). Hexadecane (0.25 ml) was used as the hydrophobic phase. The hydrophobicity was expressed as the percentage of cells adhering to the hexadecane after 15-min partitioning. Proteins were determined by the modified Lowry method (Frølund et al., 1995), using casein (Shanghai Sangon Biotechnology Co., Ltd, China) as the standard. Polysaccharides were measured by the Anthrone method (Gaudy, 1962), with glucose as the standard. The soluble chemical oxygen demand (SCOD) analyses were done using HACH DR/2000 Spectrometer (Hach Co., USA). Other sludge parameters, including total suspended solids (TSS), volatile suspended solids (VSS) and sludge volumetric index (SVI) were analyzed following the standard methods (APHA, 1998). 3. Results 3.1. Appearance and structure of stored granules
Fig. 1. The flow chart of fractionation protocol.
Original granules were both formed with a clear round outer shape (data not shown for brevity). After stored for 3 weeks, granule B remained a spherical morphotype, while granule A became irregular (Fig. 2). SEM further examined the detailed microstructures of the two stored granules (Fig. 3). It can be clearly seen that the surface of granule A had obvious crevices and pleats, and bacilli
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Fig. 2. Morphology of granules after storage. (a): granule A; (b): granule B.
Fig. 3. Scanning electron micrographs of granules after storage. (a): granule A; (b): granule B.
and cocci were found to be predominant (Fig. 3a). However, granule B (Fig. 3b) remained a compact and smooth surface on which cells were tightly attached together to form a mushroom-like structure, which would contribute to the storage stability. The comparisons of structure and appearance indicated that granules seeded with pellets were more stable than those seeded with activated sludge flocs. 3.2. Organic material concentration variations during granule storage Table 1 shows the characteristics of aerobic granules before and after storage. The SCOD of granule A and granule B increased from 181 ± 8 mg/L to 407 ± 19 mg/L and 130 ± 20 mg/L to 254 ± 11 mg/L, respectively, indicating that granule A released much more organic materials during storage. TSS and VSS dropped about 36.1% and 39.9% for granule A, while TSS and VSS of granule B only dropped about 9.4% and 14.3%, indicating a better storage stability of granule B. Both the hydrophobicity and SVI values also showed that granule B remained good settleability during storage. EPS secreted by cells were responsible for the structural integrity and could be fed as the carbon source to their own producers (Wang et al., 2007). In this study, EPS matrix was divided into Table 1 Characteristics of aerobic granules before and after storage. Parameters
SCOD (mg/L) TSS (g/L) VSS (g/L) VSS/ TSS Hydrophobicity (%) SVI (mL/g)
Before storage
After storage
Granule A
Granule B
Granule A
Granule B
181 ± 7 4.27 ± 0.35 2.63 ± 0.30 0.62 76.3 ± 5.4 47.0 ± 3.8
130 ± 20 4.49 ± 0.37 3.08 ± 0.54 0.69 85.4 ± 3.2 31.3 ± 2.6
407 ± 19 2.73 ± 0.16 1.58 ± 0.18 0.58 52.4 ± 2.6 68.4 ± 5.7
254 ± 11 4.07 ± 0.19 2.64 ± 0.19 0.65 80.6 ± 1.4 37.7 ± 2.5
supernatant, slime, LB-EPS, and TB-EPS fractions according to the fractionation procedures described in Section 2.2, and the variations of organic materials in EPS matrix are presented in Fig. 4. As shown in Fig. 4 and 81.5–96.4% of proteins and polysaccharides were presented in the TB-EPS fraction, with 2.5–8.7% of them distributed in the supernatant fraction, and less than 3.4% of them in the slime and LB-EPS fractions. Protein and polysaccharide concentrations in the supernatant for granule A were higher than those for granule B, indicating that more soluble organic materials were released to solutions for granule A during storage, which was consistent with the results of SCOD (Table 1). Furthermore, the organic materials in the TB-EPS fraction for granule A decreased much faster than those for granule B, e.g., for granule A, protein and polysaccharide concentrations decreased from 140.5 ± 4.7 mg/g VSS to 96.1 ± 6.7 mg/g VSS and 47.2 ± 0.5 mg/g VSS to 26.5 ± 0.5 mg/g VSS, whereas they did not exhibited major difference for granule B during storage. The variations of organic materials for granule A were similar to those of Wang et al. (2007), who reported that aerobic granules can degrade roughly 50% of secreted EPS when no other carbon source was available. However, the slight decline of EPS concentrations for granule B indicated that aerobic granules seeded with pellets had better storage stability. Furthermore, the proteins/polysaccharides ratio in EPS matrix for granule A decreased from the initial 4.78 to 3.78 after stored for 3 weeks, while it remained at 5.3 for granule B during the whole storage, indicating that higher proteins/polysaccharides ratio favored the storage stability. 3.3. EEM spectra in the TB-EPS fraction during storage As discussed above, most organic materials were distributed in the TB-EPS fraction and their concentration variations were related to the stability loss. In order to better characterize the organic
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Fig. 4. Variations of organic materials in EPS matrix during storage.
Fig. 5. EEM spectra in the TB-EPS fraction during storage. (A):granule A; (B):granule B.
materials in the TB-EPS fraction and explore the mechanism of stability loss, EEM fluorescence spectroscopy was applied in this study based on the principle that the fluorescence intensity was directly proportional to the organic material concentrations in lowconcentration range. As shown in Fig. 5, two main peaks could be
identified in every EEM spectra. The first peak (peak A) was located at the excitation/emission wavelengths (Ex/Em) of 220–230 nm/ 334–344 nm and reported as tryptophan protein-like material, while the second peak (peak B) was observed at the Ex/Em of 270–280 nm/340–348 nm and described as soluble microbial by-
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Fig. 6a. The outer appearances and CLSM images of original granule A. (a) green: protein (FITC); (b) violet: dead cells (SYTO blue); (c) red: nucleic acid (SYTO 63); (d) light blue: a-polysaccharide (Con A); (e) combined image of (a–d); (f) optical microscopy photograph. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6b. The outer appearances and CLSM images of original granule B. (a) green: protein (FITC); (b) violet: dead cells (SYTO blue); (c) red: nucleic acid (SYTO 63); (d) light blue: a-polysaccharide (Con A); (e) combined image of (a–d); (f) optical microscopy photograph. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
product-like material, such as aromatic protein- and humic-like material (Chen et al., 2003). Similar fluorescence signals had also
been observed in previous research (Sheng and Yu, 2006). The peak locations of granule A and granule B were almost the same, indicat-
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Fig. 7. CLSM images of stored granules. (a) green: protein (FITC); (b) light blue: a-polysaccharide (Con A); (c) red: nucleic acid (SYTO 63); (d) violet: dead cells (SYTO blue); (e) combined image of (a–d). Upper images: granule A; bottom images: granule B (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
ing the similar protein property for the two granules during storage. Thus it would not be the reason for the different protein properties but other reasons that caused the stability loss. For granule A, the SFI of peak A and peak B significantly decreased from 74.74 AU/(mg/L) DOC to 37.43 AU/(mg/L) DOC and 32.91 AU/(mg/ L) DOC to 12.64 AU/(mg/L) DOC, while the SFI for granule B did not exhibit major difference during storage. The different trends of SFIs between granule A and granule B indicated that the decline of protein concentration in the TB-EPS fraction would correspond to the stability loss of stored granules.
surface, which were consistent with the results of SEM (Fig. 3) that crevices and pleats were observed on its surface. Compared with Figs. 6 and 7, it can be easily concluded that proteins (FITC in green) were distributed throughout the original granules, while they were mainly distributed at the outer layers for stored granules. The distribution discrepancy of proteins between the original and stored granules indicated that the disintegration of protein framework caused the stability loss.
4. Discussion 3.4. CLSM images of original and stored granules In this study, multicolor fluorescence experiments are conducted to investigate the distributions of EPS and cells in the granules. Fig. 6 presents the CLSM images of original granule A and granule B, respectively, and Fig. 7 shows the CLSM images of the two stored granules. Both for the two original granules (Figs. 6a and b),1 proteins (FITC in green) and dead cells (SYTO blue in violet) were distributed throughout the entire granule, while the granule outer layer was composed of live cells (SYTO 63 in red) and a-polysaccharides (Con A in light blue). The observations suggest that proteins were generally bound to dead cell membranes, and a bioactive layer (live cells and b-polysaccharides) was located within 100 lm of the surface. The distributions of cells (live and dead) and a-polysaccharides were consistent with those of Adav et al. (2007) and Debeer et al. (1996). Moreover, this study further found that proteins were not only accumulated over the granule interior, but also spread throughout the whole granule. As shown in Fig. 7, most of EPS (protein and a-polysaccharide) and cells (total and dead) for stored granules were distributed at the outer layers, with fewer in the core region. Albeit the distribution patterns for the two stored granules were similar, their fluorescence intensities were different. The fluorescence intensity of granule A was lower than that of granule B, indicating that granule A had lower organic material concentrations in EPS matrix, which was consistent with the results of Fig. 4 that the organic materials of granule A decreased much faster than that of granule B during storage. In addition, compared with granule B, granule A had a relatively loose structure and obvious cavities were observed on the 1 For interpretation to color in Figs. 6a and b, the reader is referred to the web version of this article.
The stability loss was the most serious barrier to the practical applications of aerobic granules. Some previous publications have reported the big cavities and pleats on the surface of stored granules (Adav et al., 2008a; Wang et al., 2008). However, it appears from Figs. 2 and 3 that granules seeded with pellets remained a spherical morphotype and a compact and smooth surface. The hydrophobicity of granules was reported to decrease under the condition of carbon- and phosphorus-starvation (Wang et al., 2005, 2006). While Table 1 in this study shows that granule B, with a relatively high hydrophobicity, demonstrated a slight hydrophobicity decrease from 85% to 81% during the storage period. High hydrophobicity was beneficial to the stability, thus granules seeded with pellets were more stable than those seeded with sludge flocs. EPSs serve as the maintenance energy source during starvation, and thus contribute to the structure stability and integrity of aerobic granules (Wang et al., 2008). As can be seen in Fig. 4, more organic materials were decomposed for granule A. One important engineering implication of Fig. 4 is that when aerobic granules are subject to storage with no substrate, the reduced EPS concentration would weaken the spatial structure and the stability. The proteins/polysaccharides ratios in Fig. 4 indicated that proteins would play an important role in granules stability. The results of EEM (Fig. 5) further validated that the stability loss of stored granules was corresponded to the decrease of protein concentrations in the TB-EPS fraction. Compared with the distribution of EPS and cells in the original and stored granules (Figs. 6 and 7), the obvious difference was that proteins of the stored granules were only distributed at the outer layers, further indicating that proteins framework disintegration caused the loss of stability. While Tay et al. (2001) observed that
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the disappearance of aerobic granules was closely related to the decrease in cellular polysaccharide concentration and polysaccharides contributed to the stability of aerobic granules. The discrepancy would be due to the different analytical procedures of proteins and polysaccharides and the different EPS fractionation protocol. In industry and tourist areas, the wastewater treatment facilities sometimes have to be set into an idle phase over several days or even weeks due to intermittent wastewater production and discharge (Zhu and Wilderer, 2003). When wastewater is generated again and delivered to the treatment facilities, the activated sludge flocs may have been disintegrated already. When adopting aerobic granulation process, the granules also easily lose stability during the idle phase. Stability improvement of stored granules is therefore a challenge in the application of aerobic granules. This study showed that aerobic granules seeded with pellets were more resistant against storage than those seeded with activated sludge flocs. Based on the results obtained in this study, it appears that granules seeded with pellets would be stored and transported at room temperature, and even have the potential to be used as the seed sludge for easy and quick initiation of another aerobic reactor, which deserves further investigation. 5. Conclusion Granule B (seeded with pellets) was more resistant against storage than granule A (seeded with activated sludge flocs). An obvious decline of biomass concentration, settleability, hydrophobicity, and EPS concentration for granule A was observed when compared with granule B. The stability loss of aerobic granules was corresponded to the decrease of protein concentrations in the TB-EPS fraction and to the disintegration of protein framework in the whole granule. Aerobic granules seeded with pellets could be stored, transported and applied in some industry and tourist areas, and even applied as the seed sludge for easy and quick initiation of another aerobic reactor. Acknowledgements The authors thank the National Key Project for Water Pollution Control (2008ZX07316-002 and 2008ZX07317-003), the National Hi-Tech Research and Development Program of China (2006AA06Z384), and the Specialized Research Found for Doctoral Program of Higher Education of China (200802470029). Reference APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, American Public Health Association, Washington, DC. Adav, S.S., Lee, D.J., Tay, J.H., 2007. Activity and structure of stored aerobic granules. Environ. Technol. 28, 1227–1235.
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced storage stability of aerobic granules seeded with pellets Hua-Cheng Xu, Pin-Jing He *, Guan-Zhao Wang, Guang-Hui Yu, Li-Ming Shao State Key Laboratory of Pollution Control and Resource Reuse, Key Laboratory of Yangtze River Water Environment, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China
a r t i c l e
i n f o
Article history: Received 25 August 2009 Received in revised form 1 February 2010 Accepted 19 May 2010 Available online 17 June 2010 Keywords: Activated sludge flocs Aerobic granules Extracellular polymeric substances Pellets Storage stability
a b s t r a c t The responses of two different types of aerobic granules to storage, granule A seeded with activated sludge flocs and granule B seeded with pellets (cells), were investigated in this study. After 3-week storage, the surface of granule B remained compact and smooth while obvious crevices were observed on that of granule A. Compared with granule B, granule A had more decrease in biomass concentration, settleability, hydrophobicity, and extracellular polymeric substances (EPS) concentration after the storage. Results indicated that the stability loss of aerobic granules could be related to protein concentration decrease in the TB-EPS fraction and to protein framework disintegration in whole granule. Compared with aerobic granules seeded with activated sludge flocs, those seeded with pellets were more resistant against storage, and thus would have greater potential in practical applications. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Aerobic granulation is a novel self-immobilization process of microorganisms. Compared with conventional activated sludge, aerobic granules have the advantages of better settleability, stronger microbial structure, higher biomass retention, and better ability to handle toxic compounds (Adav et al., 2008a; Beun et al., 1999; Liu and Tay, 2002; Morgenroth et al., 1997). The application of aerobic granules was regarded as one of the promising biotechnologies in wastewater treatment (Adav et al., 2008b). Unfortunately, aerobic granules could easily lose stability and activity during storage, which would be the serious barrier to their practical application (Adav et al., 2008a; Zhang et al., 2005). Tay et al. (2002a) and Ng (2002) noted that the granules became more irregular and smaller and released soluble organic materials after storage for 8 weeks. Tay et al. (2002b) further showed that glucose-fed aerobic granules lost about 60% metabolic activity and acetate-fed granules lost about 90% metabolic activity after storing for 4 months at 4 °C in tap water. Lee et al. (2009) reported the endogenous respiration in the core of granules after starved for one month. Some storage methods were shown to improve the stability, e.g., Adav et al. (2007) proposed that storage at subfreezing temperatures ( 20 °C) was an ideal method for preserving granule stability and activity, furthermore, the addition of toxic substance (phenol) in the storing solution was beneficial to preserve the granule stability. However, this method is not quite practical be* Corresponding author. Tel./fax: +86 21 6598 6104. E-mail address: [email protected] (P.-J. He). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.05.062
cause the structure of granules would deteriorate by freezing and thawing. Although granule stability loss has been widely investigated, the information about the storage stability of aerobic granules seeded with different inocula is hardly found in the literatures so far. In most studies, aerobic granules were cultivated with activated sludge seed (Adav et al., 2008a). It is widely known that activated sludge flocs are mainly composed of cells and extracellular polymeric substances (EPS). Recently, Yu et al. (2008a, b) employed a novel EPS fractionation approach to obtain the pellets (cells) by extracting the EPS matrix. In this study, aerobic granules seeded with activated sludge flocs and pellets, respectively, were cultivated in two sequencing batch reactors (SBRs), then these two kinds of granules were both stored at 25 ± 1 °C for 3 weeks. The main purposes of this study were to investigate their responses to storage and explore the mechanism of stability loss. The EPS concentration variation during storage was monitored, and scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also applied to investigate the microstructure and distribution patterns of EPS and cells. Increased knowledge on this issue would further deepen our understanding of granule stability and be useful for the application of aerobic granules. 2. Methods 2.1. The cultivation and storage of aerobic granules Aerobic granules seeded with activated sludge flocs (granule A) and aerobic granules seeded with pellets (granule B) were
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cultivated in two SBRs, respectively. Each reactor had the same geometrical configuration with an internal diameter of 5 cm and the working H/D of 20. Fine air bubbles for aeration were introduced through porous stone-type diffuser (ACO-001, Sensen Co., Zhejiang, China) at the bottom of the column, giving a superficial gas velocity of 2.5 cm s 1. The two reactors were operated sequentially with a cycle time of 4 h (5 min of influent filling, 227 min of aeration, 3 min of settling and 5 min of effluent withdrawal). Effluent was discharged at the middle sampling port of the working column, and the hydraulic retention time (HRT) was 8 h. The reactors were housed in a temperature-controlled room at 25 ± 1 °C. The synthetic wastewater consisted of: 2000 mg/L sodium acetate; 1000 mg/L (NH4)2SO4, 200 mg/L MgCl2, 100 mg/L NaCl, 20 mg/L FeCl3, 10 mg/L CaCl2, 400 mg/L peptone, and other necessary micronutrients, and was similar to the synthetic wastewater used by Moy et al. (2002). Stable granules were both formed in the two reactors after operation for 25 days. The cultivated mature granules (named original granules) were then stored at room temperature (25 ± 1 °C) for 3 weeks with neither aeration nor nutrients. 2.2. Fractionation protocol for aerobic granules The fractionation procedures for sludge were carried out using protocols from Yu et al. (2008a, b). The flow chart of fractionation protocol is presented in Fig. 1. 2.3. Fluorescence staining and confocal laser scanning microscopy (CLSM) observation The collected original and stored granules were stained with fluorescent dyes in order to simultaneously visualize the distribution of proteins, a-polysaccharide, total cells, and dead cells in samples according to the scheme of Chen et al. (2007). The detailed approaches were: (1) Syto 63 (20 lM, 100 ll) was added to the samples, mixed on a shaker table (100 rpm) for 30 min and then washed twice with PBS solution. (2) NaHCO3 buffer (0.1 M, 100 ll) was added to maintain solution at pH 9, followed by adding a FITC solution (10 g/L, 10 ll), and the mixture was stirred for 1 h and washed twice with PBS solution. (3) The Con A solution (250 mg/L, 100 ll) was added to the samples for another 30 min and then washed twice with PBS solution. (4) The Sytox Blue solu-
tion (2.5 lM, 100 ll) was incubated with the samples for 10 min. All probes were purchased from Molecular Probes, Eugene, USA. The stained samples were frozen at 20 °C and immersed in Tissue-Tek 4583 O.C.T compound (Sakura). The 20 lm sections were then cut on a cryomicrotome (Cyrotome E, Thermo Shandon Limited, UK) for CLSM (Leica TCS SP2 confocal spectral microscope imaging system, Germany) observation. The samples were imaged with a 10 or 20 objective. 2.4. Scanning electron microscopy (SEM) observation Stored granules for SEM were: (1) Fixed with 2.5% glutaraldehyde for 2 h at room temperature. (2) Suspended in a 1% osmium tetroxide for 2 h. (3) Dehydrated via a graded series of ethanol solutions (50%, 70%, 90%, and 100%). (4) Subjected to critical point drying (Emitech, K850, UK) and coated with platinum by using a magnetron sputter coater (BEL-TEC, SCD 050 Sputter). (5) The granule structure and surface morphology were viewed via SEM (Quanta 200FEG). 2.5. Three-dimensional excitation and emission matrix (EEM) fluorescence spectroscopy The cuvettes were rinsed and ultrasonicated using 5% (w/w) nitric acid solution before analysis. All EEM spectra were measured with a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA, USA). EEM spectra were gathered with subsequent scanning emission spectra from 250 to 600 nm at 2 nm increments by varying the excitation wavelength from 200 to 500 nm at 10 nm increments. The spectra were recorded at a scan rate of 1200 nm/ min, using excitation and emission slit bandwidths of 5 nm. The voltage of the photomultiplier tube (PMT) was set to 800 V for high level light detection. Second-order Rayleigh and Raman scattering were filtered out (Lu et al., 2009). The blank scans were performed at intervals of 10 analyses using Milli-Q water. Specific fluorescence intensity (SFI) was obtained by dividing fluorescence intensity by dissolved organic carbon (DOC) concentration and reported in arbitrary units/(mg/L) DOC (AU/(mg/L) DOC). 2.6. Other analytical techniques Cell hydrophobicity was determined by the method described by Rosenberg et al. (1980). Hexadecane (0.25 ml) was used as the hydrophobic phase. The hydrophobicity was expressed as the percentage of cells adhering to the hexadecane after 15-min partitioning. Proteins were determined by the modified Lowry method (Frølund et al., 1995), using casein (Shanghai Sangon Biotechnology Co., Ltd, China) as the standard. Polysaccharides were measured by the Anthrone method (Gaudy, 1962), with glucose as the standard. The soluble chemical oxygen demand (SCOD) analyses were done using HACH DR/2000 Spectrometer (Hach Co., USA). Other sludge parameters, including total suspended solids (TSS), volatile suspended solids (VSS) and sludge volumetric index (SVI) were analyzed following the standard methods (APHA, 1998). 3. Results 3.1. Appearance and structure of stored granules
Fig. 1. The flow chart of fractionation protocol.
Original granules were both formed with a clear round outer shape (data not shown for brevity). After stored for 3 weeks, granule B remained a spherical morphotype, while granule A became irregular (Fig. 2). SEM further examined the detailed microstructures of the two stored granules (Fig. 3). It can be clearly seen that the surface of granule A had obvious crevices and pleats, and bacilli
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Fig. 2. Morphology of granules after storage. (a): granule A; (b): granule B.
Fig. 3. Scanning electron micrographs of granules after storage. (a): granule A; (b): granule B.
and cocci were found to be predominant (Fig. 3a). However, granule B (Fig. 3b) remained a compact and smooth surface on which cells were tightly attached together to form a mushroom-like structure, which would contribute to the storage stability. The comparisons of structure and appearance indicated that granules seeded with pellets were more stable than those seeded with activated sludge flocs. 3.2. Organic material concentration variations during granule storage Table 1 shows the characteristics of aerobic granules before and after storage. The SCOD of granule A and granule B increased from 181 ± 8 mg/L to 407 ± 19 mg/L and 130 ± 20 mg/L to 254 ± 11 mg/L, respectively, indicating that granule A released much more organic materials during storage. TSS and VSS dropped about 36.1% and 39.9% for granule A, while TSS and VSS of granule B only dropped about 9.4% and 14.3%, indicating a better storage stability of granule B. Both the hydrophobicity and SVI values also showed that granule B remained good settleability during storage. EPS secreted by cells were responsible for the structural integrity and could be fed as the carbon source to their own producers (Wang et al., 2007). In this study, EPS matrix was divided into Table 1 Characteristics of aerobic granules before and after storage. Parameters
SCOD (mg/L) TSS (g/L) VSS (g/L) VSS/ TSS Hydrophobicity (%) SVI (mL/g)
Before storage
After storage
Granule A
Granule B
Granule A
Granule B
181 ± 7 4.27 ± 0.35 2.63 ± 0.30 0.62 76.3 ± 5.4 47.0 ± 3.8
130 ± 20 4.49 ± 0.37 3.08 ± 0.54 0.69 85.4 ± 3.2 31.3 ± 2.6
407 ± 19 2.73 ± 0.16 1.58 ± 0.18 0.58 52.4 ± 2.6 68.4 ± 5.7
254 ± 11 4.07 ± 0.19 2.64 ± 0.19 0.65 80.6 ± 1.4 37.7 ± 2.5
supernatant, slime, LB-EPS, and TB-EPS fractions according to the fractionation procedures described in Section 2.2, and the variations of organic materials in EPS matrix are presented in Fig. 4. As shown in Fig. 4 and 81.5–96.4% of proteins and polysaccharides were presented in the TB-EPS fraction, with 2.5–8.7% of them distributed in the supernatant fraction, and less than 3.4% of them in the slime and LB-EPS fractions. Protein and polysaccharide concentrations in the supernatant for granule A were higher than those for granule B, indicating that more soluble organic materials were released to solutions for granule A during storage, which was consistent with the results of SCOD (Table 1). Furthermore, the organic materials in the TB-EPS fraction for granule A decreased much faster than those for granule B, e.g., for granule A, protein and polysaccharide concentrations decreased from 140.5 ± 4.7 mg/g VSS to 96.1 ± 6.7 mg/g VSS and 47.2 ± 0.5 mg/g VSS to 26.5 ± 0.5 mg/g VSS, whereas they did not exhibited major difference for granule B during storage. The variations of organic materials for granule A were similar to those of Wang et al. (2007), who reported that aerobic granules can degrade roughly 50% of secreted EPS when no other carbon source was available. However, the slight decline of EPS concentrations for granule B indicated that aerobic granules seeded with pellets had better storage stability. Furthermore, the proteins/polysaccharides ratio in EPS matrix for granule A decreased from the initial 4.78 to 3.78 after stored for 3 weeks, while it remained at 5.3 for granule B during the whole storage, indicating that higher proteins/polysaccharides ratio favored the storage stability. 3.3. EEM spectra in the TB-EPS fraction during storage As discussed above, most organic materials were distributed in the TB-EPS fraction and their concentration variations were related to the stability loss. In order to better characterize the organic
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Fig. 4. Variations of organic materials in EPS matrix during storage.
Fig. 5. EEM spectra in the TB-EPS fraction during storage. (A):granule A; (B):granule B.
materials in the TB-EPS fraction and explore the mechanism of stability loss, EEM fluorescence spectroscopy was applied in this study based on the principle that the fluorescence intensity was directly proportional to the organic material concentrations in lowconcentration range. As shown in Fig. 5, two main peaks could be
identified in every EEM spectra. The first peak (peak A) was located at the excitation/emission wavelengths (Ex/Em) of 220–230 nm/ 334–344 nm and reported as tryptophan protein-like material, while the second peak (peak B) was observed at the Ex/Em of 270–280 nm/340–348 nm and described as soluble microbial by-
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Fig. 6a. The outer appearances and CLSM images of original granule A. (a) green: protein (FITC); (b) violet: dead cells (SYTO blue); (c) red: nucleic acid (SYTO 63); (d) light blue: a-polysaccharide (Con A); (e) combined image of (a–d); (f) optical microscopy photograph. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6b. The outer appearances and CLSM images of original granule B. (a) green: protein (FITC); (b) violet: dead cells (SYTO blue); (c) red: nucleic acid (SYTO 63); (d) light blue: a-polysaccharide (Con A); (e) combined image of (a–d); (f) optical microscopy photograph. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
product-like material, such as aromatic protein- and humic-like material (Chen et al., 2003). Similar fluorescence signals had also
been observed in previous research (Sheng and Yu, 2006). The peak locations of granule A and granule B were almost the same, indicat-
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Fig. 7. CLSM images of stored granules. (a) green: protein (FITC); (b) light blue: a-polysaccharide (Con A); (c) red: nucleic acid (SYTO 63); (d) violet: dead cells (SYTO blue); (e) combined image of (a–d). Upper images: granule A; bottom images: granule B (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
ing the similar protein property for the two granules during storage. Thus it would not be the reason for the different protein properties but other reasons that caused the stability loss. For granule A, the SFI of peak A and peak B significantly decreased from 74.74 AU/(mg/L) DOC to 37.43 AU/(mg/L) DOC and 32.91 AU/(mg/ L) DOC to 12.64 AU/(mg/L) DOC, while the SFI for granule B did not exhibit major difference during storage. The different trends of SFIs between granule A and granule B indicated that the decline of protein concentration in the TB-EPS fraction would correspond to the stability loss of stored granules.
surface, which were consistent with the results of SEM (Fig. 3) that crevices and pleats were observed on its surface. Compared with Figs. 6 and 7, it can be easily concluded that proteins (FITC in green) were distributed throughout the original granules, while they were mainly distributed at the outer layers for stored granules. The distribution discrepancy of proteins between the original and stored granules indicated that the disintegration of protein framework caused the stability loss.
4. Discussion 3.4. CLSM images of original and stored granules In this study, multicolor fluorescence experiments are conducted to investigate the distributions of EPS and cells in the granules. Fig. 6 presents the CLSM images of original granule A and granule B, respectively, and Fig. 7 shows the CLSM images of the two stored granules. Both for the two original granules (Figs. 6a and b),1 proteins (FITC in green) and dead cells (SYTO blue in violet) were distributed throughout the entire granule, while the granule outer layer was composed of live cells (SYTO 63 in red) and a-polysaccharides (Con A in light blue). The observations suggest that proteins were generally bound to dead cell membranes, and a bioactive layer (live cells and b-polysaccharides) was located within 100 lm of the surface. The distributions of cells (live and dead) and a-polysaccharides were consistent with those of Adav et al. (2007) and Debeer et al. (1996). Moreover, this study further found that proteins were not only accumulated over the granule interior, but also spread throughout the whole granule. As shown in Fig. 7, most of EPS (protein and a-polysaccharide) and cells (total and dead) for stored granules were distributed at the outer layers, with fewer in the core region. Albeit the distribution patterns for the two stored granules were similar, their fluorescence intensities were different. The fluorescence intensity of granule A was lower than that of granule B, indicating that granule A had lower organic material concentrations in EPS matrix, which was consistent with the results of Fig. 4 that the organic materials of granule A decreased much faster than that of granule B during storage. In addition, compared with granule B, granule A had a relatively loose structure and obvious cavities were observed on the 1 For interpretation to color in Figs. 6a and b, the reader is referred to the web version of this article.
The stability loss was the most serious barrier to the practical applications of aerobic granules. Some previous publications have reported the big cavities and pleats on the surface of stored granules (Adav et al., 2008a; Wang et al., 2008). However, it appears from Figs. 2 and 3 that granules seeded with pellets remained a spherical morphotype and a compact and smooth surface. The hydrophobicity of granules was reported to decrease under the condition of carbon- and phosphorus-starvation (Wang et al., 2005, 2006). While Table 1 in this study shows that granule B, with a relatively high hydrophobicity, demonstrated a slight hydrophobicity decrease from 85% to 81% during the storage period. High hydrophobicity was beneficial to the stability, thus granules seeded with pellets were more stable than those seeded with sludge flocs. EPSs serve as the maintenance energy source during starvation, and thus contribute to the structure stability and integrity of aerobic granules (Wang et al., 2008). As can be seen in Fig. 4, more organic materials were decomposed for granule A. One important engineering implication of Fig. 4 is that when aerobic granules are subject to storage with no substrate, the reduced EPS concentration would weaken the spatial structure and the stability. The proteins/polysaccharides ratios in Fig. 4 indicated that proteins would play an important role in granules stability. The results of EEM (Fig. 5) further validated that the stability loss of stored granules was corresponded to the decrease of protein concentrations in the TB-EPS fraction. Compared with the distribution of EPS and cells in the original and stored granules (Figs. 6 and 7), the obvious difference was that proteins of the stored granules were only distributed at the outer layers, further indicating that proteins framework disintegration caused the loss of stability. While Tay et al. (2001) observed that
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the disappearance of aerobic granules was closely related to the decrease in cellular polysaccharide concentration and polysaccharides contributed to the stability of aerobic granules. The discrepancy would be due to the different analytical procedures of proteins and polysaccharides and the different EPS fractionation protocol. In industry and tourist areas, the wastewater treatment facilities sometimes have to be set into an idle phase over several days or even weeks due to intermittent wastewater production and discharge (Zhu and Wilderer, 2003). When wastewater is generated again and delivered to the treatment facilities, the activated sludge flocs may have been disintegrated already. When adopting aerobic granulation process, the granules also easily lose stability during the idle phase. Stability improvement of stored granules is therefore a challenge in the application of aerobic granules. This study showed that aerobic granules seeded with pellets were more resistant against storage than those seeded with activated sludge flocs. Based on the results obtained in this study, it appears that granules seeded with pellets would be stored and transported at room temperature, and even have the potential to be used as the seed sludge for easy and quick initiation of another aerobic reactor, which deserves further investigation. 5. Conclusion Granule B (seeded with pellets) was more resistant against storage than granule A (seeded with activated sludge flocs). An obvious decline of biomass concentration, settleability, hydrophobicity, and EPS concentration for granule A was observed when compared with granule B. The stability loss of aerobic granules was corresponded to the decrease of protein concentrations in the TB-EPS fraction and to the disintegration of protein framework in the whole granule. Aerobic granules seeded with pellets could be stored, transported and applied in some industry and tourist areas, and even applied as the seed sludge for easy and quick initiation of another aerobic reactor. Acknowledgements The authors thank the National Key Project for Water Pollution Control (2008ZX07316-002 and 2008ZX07317-003), the National Hi-Tech Research and Development Program of China (2006AA06Z384), and the Specialized Research Found for Doctoral Program of Higher Education of China (200802470029). Reference APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, American Public Health Association, Washington, DC. Adav, S.S., Lee, D.J., Tay, J.H., 2007. Activity and structure of stored aerobic granules. Environ. Technol. 28, 1227–1235.
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