Effects of activated sludge flocs and pellets seeds on aerobic granule properties

Journal of Environmental Sciences 2011, 23(4) 537–544

Effects of activated sludge flocs and pellets seeds on aerobic granule properties Huacheng Xu, Pinjing He ∗, Guanzhao Wang, Liming 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, China. E-mail: [email protected] Received 23 May 2010; revised 05 July 2010; accepted 12 July 2010

Abstract Aerobic granules seeded with activated sludge flocs and pellets (obtained from activated sludge flocs) were cultivated in two sequencing batch reactors and their characteristics were compared. Compared with granules seeded with activated sludge flocs, those seeded with pellets had shorter start-up time, larger diameter, better chemical oxygen demand removal efficiency, and higher hydrophobicity, suspended solid concentration, and Mg2+ content. The different inocula led the granule surface with different microbial morphologies, but did not result in different distribution patterns of extracellular polymeric substances and cells. The anaerobic bacterium Anoxybacillus sp. was detected in the granules seeded with pellets. These results highlighted the advantage of pellet over activated sludge floc as the seed for aerobic granulation and wastewater treatment. Key words: activated sludge flocs; aerobic granule; comparison; pellets; sequencing batch reactor DOI: 10.1016/S1001-0742(10)60445-7 Citation: Xu H C, He P J, Wang G Z, Shao L M, 2011. Effects of activated sludge flocs and pellets seeds on aerobic granule properties. Journal of Environmental Sciences, 23(4): 537–544

Introduction Aerobic granulation has been widely investigated in sequencing batch reactors (SBRs) as a novel environmental biotechnology. Compared with activated sludge flocs, aerobic granules have the advantages of compact and strong microbial structure, good settling ability, high biomass retention, and ability to withstand high organic loading rate (Adav et al., 2008; Beun et al., 1999; Liu and Tay, 2002). Nowadays, aerobic granules have been successfully applied for the treatment of high strength organic wastewater (Moy et al., 2002), toxic organic wastewater (Jiang et al., 2002), heavy metals and dyes (Xu et al., 2004), dairy and brewery wastewater (Schwarzenbeck et al., 2005; Wang et al., 2007), and even low-strength domestic wastewater (Wang et al., 2009). Aerobic granulation is affected by many operational parameters, such as organic loading rate, carbon source, hydrodynamic shear force, cycle/settling time, volume exchange ratio, and extracellular polymeric substance (EPS) (Liu and Tay, 2004). Although aerobic granulation has been investigated extensively during the last 20 years, most aerobic granules cultivated in previous research were seeded with activated sludge flocs, and the effects of different inocula on aerobic granulation have not yet been fully investigated. The bacterial community in activated seed sludge is * Corresponding author. E-mail: [email protected]

important for aerobic granulation as hydrophobic bacteria more likely attaches to sludge flocs than its hydrophilic counterpart (Zita and Hermansson, 1997). EPS in activated sludge flocs mainly consists of polymeric carbohydrates, proteins, lipids, DNA, and is considered hydrophilic (Schmidt and Ahring, 1994). Thus, it is reasonable to conclude that EPS would play a negative role in the initial aerobic granulation. The EPS can be extracted using both physical and chemical approaches (Gessesse et al., 2003; Li and Yang, 2007; Zhang et al., 2007). A novel EPS fractionation approach was employed in our previous studies to obtain the pellets by extracting the EPS matrix (Shao et al., 2009; Yu et al., 2008a). In the present study, the above-mentioned pellets were applied as inocula to cultivate aerobic granules using acetate as the sole carbon and energy source. Activated sludge flocs sampled from a local wastewater treatment plant (WWTP) were also employed to cultivate aerobic granules as the control. The main purpose of this article was to investigate the effects of different inocula on aerobic granulation process and granule characteristics. Particle size distribution (PSD), the organic materials in EPS matrix, scanning electron microscopy (SEM) images, confocal laser scanning microscopy (CLSM), and denaturing gradient gel electrophoresis (DGGE) fingerprint profile were applied to compare their characteristics. This work offers a detailed new operation strategy for aerobic granulation and could be useful for pilot- and full-scale application.

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1 Materials and methods 1.1 Granule cultivation Two column-type reactors (R1 seeded with activated sludge flocs and R2 seeded with pellets) with a working volume of 2.0 L were used as SBR reactors to cultivate aerobic granules. Each reactor had an internal diameter of 5 cm and the height of 100 cm. Fine aeration air bubbles were introduced through porous a stone-type diffuser at the bottom of the column, giving a superficial gas velocity of 2.5 cm/sec. During aeration, the airflow rate was 3.0 L/min and the dissolved oxygen was maintained above 2.5 mg/L. The reactors were operated sequentially with a cycle time of 4 hr (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 column, and the hydraulic retention time was 8 hr. The reactors were housed in a temperature-controlled room at (25 ± 1)°C. Synthetic wastewater with acetate as the sole carbon source was used in this work, with the following composition (mg/L): sodium acetate 2000, (NH4 )2 SO4 1000, MgCl2 200, NaCl 100, FeCl3 20, CaCl2 10, peptone 400; phosphate buffer (3350 mg/L KH2 PO4 , 4100 mg/L K2 HPO4 ); and micronutrients (mg/L): H3 BO3 50, ZnCl2 50, CuCl2 30, MnSO4 ·H2 O 50, (NH4 )6 Mo7 O24 ·4H2 O 50, AlCl3 50, CoCl2 ·6H2 O 50, and NiCl 50 (Moy et al., 2002). This gave a loading rate of 4.95 kg chemical oxygen demand (COD)/(m3 ·day). 1.2 Fractionation protocol and EPS The procedures for EPS fractionation were modified from previous research (Shao et al., 2009; Yu et al., 2008a). In brief, the sludge sample was first settled for 1.5 hr at 4°C, the bulk solution (supernatant) was collected carefully. The sediments were collected and re-suspended to their original volumes using a phosphate buffered saline (PBS) solution and then centrifuged at 2000 ×g for 15 min, and the bulk solution was collected as the slime, representing the fraction that could be removed by soft centrifugation. The bottom sediments were then re-suspended to their original volumes with the PBS solution and centrifuged at 5000 ×g for 15 min, and the organic materials in the supernatant were the LB-EPS. The collected sediments were re-suspended again with the PBS buffer solution to original volumes and extracted using ultrasound at 20 kHz and 480 W for 10 min. The extracted suspensions were centrifuged at 20,000 ×g for 20 min. The organic materials in the bulk solution were the TB-EPS, while the residues (solid phase) re-suspended with the PBS solution to original volumes were the pellets. Hence, from the surface to the core of the granule, the sludge sample possessed a multi-fractioned structure consisting of supernatant, slime, LB-EPS, TBEPS, and pellet fractions. 1.3 SEM and CLSM observations Aerobic granules for SEM were first fixed with 2.5% glutaraldehyde for 2 hr at room temperature. Then, the granules were suspended in a 1% osmium tetroxide for an-

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other 2 hr. Subsequently, the granules were dehydrated via a graded series of ethanol solutions (50%, 70%, 90%, and 100%), subjected to critical point drying (K850, Emitech, UK), and coated with platinum using a magnetron sputter coater (BEL-TEC, SCD 050 Sputter, Switzerland). Finally, the granule structure and surface morphology were viewed via SEM (Quanta 200FEG, USA). Before CLSM observation, the collected granules were stained with fluorescent dyes to simultaneously visualize the distribution of proteins, α-polysaccharide, total cells, and dead cells (Chen et al., 2007). Specifically, 20 μmol/L Syto 63 (100 μL) was initially added to the samples on a shaker table (100 r/min) for 30 min. Then, 0.1 mol/L NaHCO3 buffer (100 μL) was added to maintain the solution at pH 9, followed by adding a FITC solution (10 g/L, 10 μL), and the mixture was stirred for 1 hr. Next, 250 mg/L Con A solution (100 μL) was added to the samples for another 30 min. Samples were washed twice with PBS solution to remove the extra dyes for each of the abovementioned three staining stages. Finally, 2.5 μmol/L Sytox Blue solution (100 μL) 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-μm 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 20× objective. 1.4 DNA extraction and DGGE The procedures for DNA extraction were carried out using protocols described by Ye et al. (2007). Primer 338f with GC clamp and 518r were used to amplify the V3 region of 16S ribosomal DNA (16S rDNA) genes from the bacterial community. The polymerase chain reaction (PCR) amplification was performed using 50 μL mixture containing 1 μL primer GC-338f (10 pmol/L), 1 μL primer 518r (10 pmol/L), 1 μL dNTP (100 μmol/L), 5 μL PCR buffer (10×), 4 μL MgCl2 (20 mmol/L), 0.25 μL Taq polymerase, 2 μL DNA extract, and 36.75 μL H2 O. The PCR recycling was performed as follows: one cycle of 94°C for 2 min, 30 cycles of denaturation at 94°C for 45 sec, anneal at 60°C for 45 sec, extension at 72°C for 90 sec, and a single final extension at 72°C for 10 min. The PCR product was around 230 bp in length. The DGGE was performed with the DCodeTM Universal Detection System according to the manufacturer’s instructions (BioRad Laboratories, USA). Forty microlitres of PCR product was loaded onto 8% polyacrylamide gels with denaturing gradients ranging from 30% to 55% (where 100% denaturant contains 7 mol/L urea and 40% formamide) in 1× TAE buffer. The electrophoresis was run at a constant voltage of 150 V at 60°C for 4 hr. After that, the gel was stained with ethidium bromide and photographed. Small pieces of selected DGGE bands were excised from the DGGE gel and rinsed in 1 mL deionized water. The gel was then crushed in 200 μL of sterile deionized water, incubated at

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2 Results and discussion

30°C for 1 hr, and stored at –20°C. 1.5 Other analytical techniques

2.1 Granule formation and morphology

The PSD assay was determined by a Mastersizer 2000 (Malvern, UK) which enabled the measurement of particles in the range 0.1–2000 μm. The samples were diluted in filtrated effluent by a 0.45 μm polytetrafluoroethylene (PTFE) membrane to avoid multiple scattering. Each sample was gently taken by a wide mouth pipet. Hydrophobicity was determined by the method described by Rosenberg et al. (1980). Hexadecane (0.25 mL) was used as the hydrophobic phase, and the hydrophobicity was expressed as the percentage of cells adhering to the hexadecane after 15-min partitioning. Cation concentration in the EPS matrix was determined by inductively coupled plasma-atomic emission spectrometry (ICP-optima 2001DV, PerkinElmer, USA). 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 COD was determined using a HACH DR/2000 Spectrometer (Hach Company, USA). Other sludge parameters, including total suspended solids (TSS) and volatile suspended solids (VSS), were analyzed following the standard methods (APHA, 1998).

The formation of aerobic granulation is a gradual process involving progression from seed sludge to compact aggregates and finally to compact mature granules. One characteristic of aerobic granules compared with activated sludge flocs is their relatively large particle size. Figure 1 illustrates the changes of particle sizes in aerobic granulation. The activated sludge flocs taken from WWTP were grayish brown and showed a fluffy, irregular and loosestructural morphology (photos not shown). Their color changed from brown to yellow by the end of the experimental process. A similar tendency of color variation was also observed by Wang et al. (2007). The initial particle size in R2 (24 μm) was smaller than those in R1 (85 μm), which was due to the sludge disintegration caused by ultrasound extraction (Wang et al., 2005; Yu et al., 2008b). After startup, the particles in R2 increased so fast that the two reactors almost had the same diameters (170–180 μm) after three days of operation, suggesting that pellets were beneficial for the initial formation of aggregates. Aerobic granules were firstly observed in R2 after 10 days of operation, while the same phenomenon was observed in R1 at day 15. These observations clearly showed that the application of pellets accelerated granulation, which was likely related to the elimination of EPS steric interference (Yu et al., 2009). Mature granules dominated in both reactors after 30 days cultivation, while granules in R2

100 Day 1

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Changes of particle sizes in aerobic granulation. R1 was seeded with activated sludge flocs, and R2 was seeded with pellets.

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5

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Time (day) Total suspended solid (TSS), volatile suspended solids (VSS) and effluent COD concentrations in aerobic granulation.

were larger (3.5 mm) than those in R1 (2.1 mm), showing the application of pellets seed could also lead to larger mature granules. 2.2 Granule property and reactor performances Figure 2 shows the changes of TSS, VSS and COD removal in aerobic granulation. The TSS concentration in R1 decreased from 0.63 to 0.15 g/L at day 3 due to the washout of the light and dispersed flocs from the reactors in the initial period of operation, then increased steadily and reached (4.27 ± 0.35) g/L after 30 days of operation. The evolutions of TSS and VSS in R2 were similar with those in R1, but the final TSS concentration in R2 was (4.49 ± 0.37) g/L, being higher than in R1. The application of pellets resulted in higher biomass concentration. The reactor performance, in terms of effluent COD concentration, exhibited a decreasing trend in the two reactors. The COD concentration in the influent was 1650 mg/L, after 30 days of operation, and the COD in the effluent of R1 and R2 reached (181 ± 7) mg/L and (130 ± 20) mg/L, respectively, implying that R2 had the higher COD removal efficiency (92.7%) than R1 (89.0%). The higher COD removal efficiency in R2 was consistent with the higher biomass concentrations mentioned above. The hydrophobicity of the two inocula was less than 50%, but by the end of the experiment the hydrophobicity of granules in R1 and R2 increased to 76.3% ± 5.4% and 85.4% ± 3.2%, respectively (data not shown). This showed that hydrophobicity of aerobic granules was nearly 50%– 70% higher than that of the seed sludge and the application of pellets was associated with higher hydrophobicity. High hydrophobicity favored the formation of aerobic granules (Liu and Tay, 2002), which was responsible for the acceleration of aerobic granulation in R2. 2.3 Variations of proteins and polysaccharides in aerobic granulation As formation of aerobic granules is associated with organic materials (McSwain et al., 2005; Tay et al., 2001), we investigated the variations of organic materials in EPS matrix in aerobic granulation. As shown in Fig. 3, proteins and polysaccharides were mainly distributed in the TB-EPS fraction, less distributed in the supernatant, and almost not found in the slime and LB-EPS fractions. It

was reasonable to conclude, therefore, that the formation of aerobic granules was mainly correlated with organic materials in the TB-EPS fraction. Thus, the variations of proteins and polysaccharides in the TB-EPS fraction are important. As for R1, the protein concentration in the TB-EPS fraction (Fig. 3a) changed slightly during the first 20 days, followed by a sharp increase and reached (140.5 ± 4.7) mg/g VSS on day 30, while the polysaccharide concentration (Fig. 3c) did not exhibit a major difference and stabilized at about 30 mg/g VSS during the whole aerobic granulation. Interestingly, the protein and polysaccharide concentrations in the TB-EPS fraction for R2 (Fig. 3b and 3d) sharply increased after startup, indicating that more EPSs were secreted during the initial granulation. Afterwards, the protein concentration decreased to (70.2 ± 0.8) mg/g VSS on day 15, followed by an increase to (150.1 ± 5.0) mg/g VSS at the end of experiment, while polysaccharide concentration gradually decreased to (28.4 ± 2.0) mg/g VSS. The application of different inocula resulted in a difference in EPS distribution patterns, especially at the initial granulation stage. The concentrations of proteins and polysaccharides in the supernatant for the two reactors both increased sharply during the first 10 days, followed by a decline in the subsequent operation time. The initial increase was due to the incompact structure and the release of organic materials caused by strong hydraulic shear forces. After aerobic granules were formed with compact structure, the organic materials were then transferred from the supernatant to the TB-EPS fraction. The ratios of proteins to polysaccharides of mature granules in R1 and R2 were 3.00 and 5.04 respectively, both higher than those of the inocula (1.82). This phenomenon indicated that the application of pellets seed resulted in higher protein to polysaccharide ratios. McSwain et al. (2005) also reported high protein concentration (proteins to polysaccharide ratios of 6.6–10.9) in aerobic granules. While Tay et al. (2001) reported that hydrodynamic shear increased intracellular polysaccharide production. Claudio et al. (2006) even noted that hydrodynamic shear facilitated the formation of granules but did not affect the EPS concentration and composition. These inconsistent conclusions are likely due to the different EPS fraction procedures

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d

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Polysaccharide (mg/g VSS)

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0

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541

40 30 20 10

40 30 20 10

0 Supernatant Slime LB-EPS TB-EPS Slime LB-EPS TB-EPS EPS fraction EPS fraction Fig. 3 Variations of proteins and polysaccharides in aerobic granulation. (a) proteins in R1; (b) proteins in R2; (c) polysaccharides in R1; (d) polysaccharides in R2. 0

Supernatant

and analytical methods. The present study demonstrated that proteins, rather than polysaccharides, were enriched in the granules, which was consistent with the results of McSwain et al. (2005).

with pellets had larger diameter, shorter start-up time, and higher biomass concentration. More detailed CLSM images of aerobic granules can be found elsewhere (Xu et al., 2010).

2.4 SEM and CLSM observations

2.5 Variations of cation contents in EPS matrix in aerobic granulation

To understand the detailed microstructures, SEM was performed on two granules. As shown in Fig. 4a and c, both granules had a clear, compact physical structure. The SEM images at high magnification showed that different microbial clusters were dominated on R1 and R2 surfaces. Filamentous cells and rod-shaped bacteria were found on the R1 surface (Fig. 4b), while granules cultivated in R2 were populated with nonfilamentous bacteria, and showed a compact and smooth surface on which cells were tightly attached to form a mushroom-like structure (Fig. 4d). Li et al. (2009) also observed the appearance of various rodshaped bacteria on granule surfaces, which was similar to the surfaces of granules cultivated in R1. The CLSM results showed that aerobic granules cultivated in R1 and R2 had similar EPS and cell distribution patterns. More specifically, proteins and dead cells were distributed throughout the entire granule, while the outer layer was composed of live cells and α-polysaccharides. The CLSM observation indicated that the application of pellets seed did not result in different EPS and cell distribution patterns, despite the fact that granules seeded

We investigated cations due to their high association with organic materials in sludge flocs (Novak et al., 2007), and a distinct distribution pattern in the different EPS fractions was observed (Fig. 5). For activated sludge flocs, 74.1% of Ca2+ and 73.1 % of Mg2+ were distributed in the supernatant and slime fractions, whereas 89.4% of Fe3+ and 81.8 % of Al3+ were presented in the TB-EPS fraction. That is, bivalent cations were mainly distributed in the outer fractions (supernatant and slime), and trivalent cations were principally presented in the inner fraction (TB-EPS). The obvious variation in aerobic granulation was the increasing Mg2+ content, suggesting that the formation of aerobic granules in this study may depend on Mg2+ accumulation. As for R1, Mg2+ content increased by 457%, 10%, 47%, and 116% in the supernatant, slime, LB-EPS, and TB-EPS fractions, respectively. While for R2, it increased by 475%, 50%, 79%, and 164% in the supernatant, slime, LB-EPS, and TB-EPS fractions, respectively. It is likely that the application of pellets seed caused a greater increase in Mg2+ content. Divalent

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Fig. 4 Scanning electron microscopy images of the aerobic granule after 30 days of operation. (a) R1 (×150); (b) R1 (×30,000); (c) R2 (×150); (d) R2 (×20,000).

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Al Ca Mg Fe

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Slime LB-EPS EPS fraction Fig. 5 Variations of cation contents in EPS matrix in aerobic granulation.

TB-EPS

Table 1 Sequence analysis and species identification for reactors R1 and R2 Band No.

Sequence (bp)

Closest bacterial sequence (Gen bank accession no.)

Identity (%)

1 2 3 4 5 6 √

157 151 165 152 153 159

Brevibacillus sp. 3n (FJ490610.1) Uncultured bacterium (EU426931.1) Comamonadaceae bacterium (DQ066969.1) Comamonas sp. PD-13u (AB195166.1) Anoxybacillus sp. B1 (FJ268956.1) Ureibacillus sp. A3.03 (EF105478.1)

94 96 95 89 97 98

R1 √ √ √ √ × √

R2 √ √ √ √ √ √

: strains detected; ×: strains not detected.

metal ions, such as Ca2+ , enhance granulation (Yu et al., 2001; Jiang et al., 2003). We found, however, that Mg2+

rather than Ca2+ was the essential element for aerobic granulation. The results of the present study are similar

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flocs, those seeded with pellets had larger diameter, more biomass concentration, better COD removal efficiency, and higher hydrophobicity and Mg2+ content. Additionally, anaerobic bacterium Anoxybacillus sp. was detected in the granules seeded with pellets. The application of pellets offers a new operation strategy for starting up aerobic granulation systems and for pilot- and full-scale application.

R2

d0 d5 d10 d15 d20 d30 d0 d5 d10 d15 d20 d30

Acknowledgments

Band 1 Band 2 Band 3 Band 4 Band 5 Band 6

This work was supported by the National Natural Science Foundation of China (No. 20977066), the National Key Project for Water Pollution Control (No. 2008ZX07316-002, 2008ZX07317-003), and the Specialized Research Fund for Doctoral Program of Higher Education of China (No. 200802470029).

References

Fig. 6 DGGE profiles of the bacterial communities in aerobic granulation.

to that of Li et al. (2009) who also reported that Mg2+ enhanced granulation. We surmised that magnesium linked with EPS and formed an EPS-Mg2+ -EPS cross linkage. 2.6 Microbial analysis in aerobic granulation Figure 6 represents the DGGE fingerprint profile of PCR amplified sequences for R1 and R2 in aerobic granulation. Each DGGE band was different from others during the experiment, indicating that the structure of the microbial communities was dynamic rather than static in aerobic granulation. The bacterial populations in the two reactors changed at different rates, which resulted in different DGGE bands and patterns. The number of DGGE bands in R1 decreased at the initial granulation stage (first 10 days), increased as small granules formed (day 15), decreased again (day 20), and finally increased as mature aerobic granules were formed (day 30). More DGGE bands were observed in R2 than in R1, indicating that R2 had a larger microbial community. More importantly, compared with the microbial community detected in R1, Anoxybacillus sp. (band 5) was identified in R2 (Table 1). Anoxybacillus sp. is an anaerobic bacterium (Pikuta et al., 2000), and its appearance may indicate the existence of an anaerobic environment in the internal core. Thus, compared with aerobic granules seeded with activated flocs, those seeded with pellets may have the potential for nitrogen and phosphorus removal, which requires further investigation.

3 Conclusions Pellets were more appropriate inocula than activated sludge flocs for aerobic granulation. Compared with aerobic granules seeded with conventional activated sludge

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