Stable aerobic granules for continuous-flow reactors: Precipitating calcium and iron salts in granular interiors

Bioresource Technology 101 (2010) 8051–8057

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Stable aerobic granules for continuous-flow reactors: Precipitating calcium and iron salts in granular interiors Yu-Chuan Juang a, Sunil S. Adav a, Duu-Jong Lee a,*, Joo-Hwa Tay b a b

Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan Department of Environmental Science and Engineering, Fudan University, Shanghai, China

a r t i c l e

i n f o

Article history: Received 10 February 2010 Received in revised form 22 May 2010 Accepted 25 May 2010 Available online 19 June 2010 Keywords: Aerobic granule Continuous-flow reactor Stability Calcium Precipitate

a b s t r a c t Aerobic sludge granules are compact, strong microbial aggregates that have excellent settling ability and capability to efficiently treat high-strength and toxic wastewaters. The aerobic granules cultivated with low ammonium and phosphates lost structural stability within 3 days in continuous-flow reactors. Conversely, stable aerobic granules were cultivated in substrate with high levels of ammonium salts that could stably exist for 216 days in continuous-flow reactors with or without submerged membrane. The scanning electron microscopy, energy dispersive spectroscopy microanalysis and the confocal laser scanning microscopy imaging detected large amounts of calcium and iron precipitates in granule interiors. The Visual MINTEQ version 2.61 calculation showed that the phosphates and hydroxides were the main species in the precipitate. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Aerobic granular sludge formed by self-immobilized microbial cells has high settling velocity and high biomass retention in a compact sequencing batch reactor (SBR) (Morgenroth et al., 1997; Beun et al., 1999; Tay et al., 2001; Yang et al., 2003; Liu and Tay, 2004; Adav et al., 2007a, 2008a). In response to these unique granule attributes, aerobic granulation technology was developed for treating high-strength wastewater containing organic compounds, nitrogen, phosphorus, toxic substances and xenobiotics (Jiang et al., 2002; Moy et al., 2002; Tay et al., 2002; Adav et al., 2007b; Adav and Lee, 2008). Aerobic granules were successfully cultivated in an SBR with short settling time and appropriate feast/famine ratios. Aerobic granular sludge combined with the membrane bioreactor process, the aerobic granule membrane bioreactor (AGMBR) process, was studied (Jun et al., 2007; Li et al., 2007; Tay et al., 2008; Juang et al., 2008). During long-term SBR operation, the aerobic granular sludge process gradually loses stability under inappropriate operational conditions (Li et al., 2007; Liu and Liu, 2006; Adav et al., 2008b; Zheng et al., 2006). Steady-state continuous-flow reactors are preferred over SBRs as the former has lower installation cost, and easy operation, maintenance, and control. Experimental results demonstrate that aerobic granules in a continuous-flow reactor lose stability faster than in an SBR (Chen et al., 2009). Granules stability * Corresponding author. Tel.: +886 2 33663028; fax: +886 2 23623040. E-mail address: [email protected] (D.-J. Lee). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.05.078

can only be cultivated and maintained for a period in an SBR. However, aerobic granules that can stably exist over long-term, continuous-flow operation are highly desirable. Although aerobic granules can be cultivated at different organic loading and reactor operation conditions, they lose stability during long term reactor operation suggesting that the self-immobilized, compact state is not thermodynamically favourable for the constituent cells. This study cultivated aerobic granules with long term stability in continuous-flow reactors with enriched denitrifier community and high concentrations of phosphate salts. The seed granules from an SBR remained structurally stable and maintained a satisfactory organic degradation rate during 216-d test in continuous-flow reactors. 2. Methods 2.1. Cultivation of seed aerobic granules in SBR Activated sludge obtained from a local municipal wastewater treatment plant in Taipei, Taiwan, was inoculated in column-type SBR (120  6 cm). The reactor was fed with synthetic wastewater containing acetate as the sole carbon source with the following media (in g l1): 1.0 (NH4)2SO4; 0.2 NaCl; 0.2 MgSO47H2O; 0.02 FeCl3; 0.01 CaCl22H2O; 1.65 K2HPO4; 1.35 KH2PO4; and micronutrients 1.0 ml l1 (Adav et al., 2007b). The initial organic loading rate was 1.7 kg COD m3, and was step increased to 16.7 kg COD m3 d1 by proportionally adjusting the concentration of each chemical ingredient, except for that of buffer constitutes (K2HPO4

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and KH2PO4). Fine air bubbles for aeration and mixing were fed through the reactor bottom at a superficial velocity of 3.4 cm s1. The reactor was operated sequentially in 4-h cycles with 5 min settling time and 10 min effluent withdrawal; the remaining time in each cycle was reaction time. The volumetric exchange ratio of liquid was 50%. The mature granules with COD removal potentials of 95–96% at 16.7 kg COD m3 d1 were sampled after a 60-d cultivation phase. These granules, termed herein as ‘‘seed granules I”, were seed granules for the continuous-flow reactor. In control tests, granules were cultivated using the same SBR and the same synthetic wastewater mentioned above but with much lower phosphate salts (in g l1): 0.1 K2HPO4 and 1.35 KH2PO4. Mature granules, termed herein as ‘‘seed granules II”, were also successfully cultivated in 60 d with high COD removal efficiency. However, the granule is of a puffy surface with filamentous bacteria growth. 2.2. Continuous-flow reactor set-up and operation Two identical laboratory-scale column-type (120  6 cm) continuous-flow reactors were utilized in this study. Seed granules I or seed granules II were fed into the continuous-flow reactors for long term operation test. Synthetic wastewater for continuousflow reactor tests had the same composition as that in the granule cultivation phase (Section 2.1 in an SBR), except that the acetate concentration was fixed to generate an organic loading rate (OLR) of 7.0 kg COD m3 d1. In reactor A, synthetic wastewater was fed continuously and effluent was over-flown out through a top weir, yielding a hydraulic retention time (HRT) of 24 h. Coarse bubbles were generated at a flow rate of 3 l min1 through a bottom diffuser. All operational parameters for reactor B were the same as those for Reactor A, except that a 0.03 m2 membrane module made of polyethylene hollow-fibre membranes 500 lm diameter with 0.4 lm pores (Mitsubishi Rayon Co., Tokyo, Japan), was installed above the bottom gas diffuser. 2.3. Granule staining and scanning Seed granules I from the SBR and granule samples from the continuous-flow reactors were collected and maintained fully hydrated during staining. The SYTO 63 dye, which is a cell-wallpermeable nucleic acid stain, was used to stain total cells. The SYTOX blue dye, a cell-wall-impermeable stain, was used to stain dead cells in granules. The Calcium Green dye was employed to stain incorporated calcium ions. Granule samples were washed with Milli-Q water and then with phosphate-buffered saline (PBS) (100 mM, pH 7.4). The SYTO 63 (20 lM and 100 lL) was first dripped onto the sample, placed on a shaker table for 30 min, followed by SYTOX Blue (2.5 lM and 100 lL) dripping for 30 min. The sample was then stained with Calcium Green (10 lM and 100 lL) for 30 min. After each staining stage, samples were washed twice with PBS buffer to remove excess stain. All probes were purchased from Molecular Probes (CA, USA). Confocal laser scanning microscopy (CLSM) (Leica TCS SP5; Confocal Spectral Microscope Imaging System, GmbH, Wetzlar, Germany) was employed to examine the distribution of total cells, dead cells and calcium in the granule interior. Granule samples were imaged using 10 or 20 objectives; images were analyzed using Leica confocal software (Leica TCS SP2; Confocal Spectral Microscope Imaging System, GmbH, Wetzlar, Germany). The fluorescence of SYTO 63 (red) was determined via excitation at 633 nm and emission at 650–700 nm. Fluorescent intensity of SYTOX Blue (pink) was detected via excitation at 458 nm and emission at 460–500 nm. Excitation and emission wavelengths were set at 488 nm and 500–580 nm for detection of Calcium Green

(green). The general staining procedures are described by Chen et al. (2006) and Yang et al. (2009). 2.4. DNA isolation, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) The DNA from the reactor samples collected at particular interval during reactor operation period were extracted via enzymatic lysis using extraction buffer (100 mM Tris–HCl at pH 8.0, 100 mM EDTA at pH 8.0, and 1.5 M NaCl) containing Proteinase K (10 mg ml1) (Amresco Inc., Solon, OH, USA), as described earlier (Adav et al., 2007b). Polymerase chain reaction (PCR) amplification of the 16S rRNA gene was conducted using extracted DNA with primer P2 and primer P3. The nucleotide sequences of the primers were P1 (50 -CCTACGGGAGGCAGCAG-30 ), P2, (50 -ATTACCGC GGCTGCTGG-30 ) and P3 (50 -CGCCCGCCGCGCGCGGCGGGCGGG GCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-30 ). Primer P3 contains the same sequence as primer P1 with additional 40-nucleotide GC-rich sequence (GC clamp) at its 50 end (Muyzer et al., 1993). The 50 ll PCR mixture consisted of 5 ll PCR buffer (10), 2.5 ll MgCl2 (25 mM), 2.5 ll dNTP (2.5 mM), 1.0 ll (10 mM) each of the primer, 0.5 ll Taq polymerase (Promega, Madison, WI, USA) and 100 ng of the DNA template. The PCR-amplification was performed using an eppendorf mastercycler (Eppendorf AG, Hamburg, Germany) by denaturation at 94 °C for 3 min and 25 cycles consisting of 94 °C for 30 s, 55 °C for 60 s, 72 °C for 90 s, and final extension at 72 °C for 7 min. Denaturing gradient gel electrophoresis (DGGE) tests were conducted using Bio-Rad universal mutation detection system with 10% (w/v) polyacrylamide gels. The range of denaturants (100% denaturant corresponds to 7 M urea and 40% (v/ v) deionized formamide) was 35–65%. The DGGE was performed at 60 °C for 18 h at 100 V. Gels were stained with ethidium bromide and photographed using a UV transilluminator. The structural diversity of the microbial community was deterP mined using the Shannon index, calculated by H ¼  Pi log P i , where Pi is the importance probability of bands in the gel lane. The species richness was calculated by d ¼ ðS  1Þ=logðNÞ. The Bray–Curtis similarity indices were employed to compare structural communities based on the DGGE banding profiles of samples using Primer software. 2.5. Construction of a clone library and analysis of microbial diversity in the AGMBR The DNA from AGMBR granules were extracted via enzymatic lysis using extraction buffer (100 mM Tris–HCl at pH 8.0; 100 mM EDTA at pH 8.0; and 1.5 M NaCl) containing Proteinase K (10 mg ml1) (Amresco, Inc., Solon, OH, USA), as described previously (Adav et al., 2007b). Isolated DNA was purified by GeneSpin™ (Protect Technology Co. Ltd., USA). The PCR amplification of 16S rRNA gene sequences utilized primers F27 (50 -CCA GAG TTT GAT CMT GGC TCA G-30 ) and R1492 (50 -TAC CTT GTT ACG ACT T-30 ). The PCR amplification was conducted using 50 ll reaction volumes containing PCR buffer with 1.5 mM MgCl2, 200 lM dNTPs, 100 ng genomic DNA, 0.5 U Taq DNA polymerase, and1 lM of each primer. The PCR amplification was performed in an Eppendorf mastercycler (Eppendorf AG, Hamburg, Germany) under the following conditions: 1 cycle at 95 °C for 3 min; 25 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s; and 1 10-min cycle at 72 °C. The PCR products were purified, cloned using the QIAGEN PCR cloning kit (QIAGEN, Valencia, CA, USA) as per the manufacturer’s instructions, and transformed into E. coli TOP10F cells (Invitrogen, Carlsbad, CA, USA). Fifty clones were randomly selected and sequenced using the ABI Prism model 3730 version 3.2. The 16S rRNA gene sequences were aligned using the multiple alignments CLUSTAL W. The phylogenetic trees-based 16S rRNA

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tents of samples were determined using the Anthrone method (Gaudy, 1962) with glucose as the standard. In brief, 0.1 ml of the sample was added into 5 ml of anthrone reagent and test tubes were immediately kept in boiling water bath for 10 min. After cooling to room temperature, absorbance was measured at 620 nm. The blank test contained 0.1 ml deionised water and anthrone reagent. The PN contents were identified using the modified Lowry method (Frolund et al., 1995) with bovine serum albumin as the standard. In short, 0.2 ml test sample was incubated with 1 ml Lowry reagent for 10 min. Then 0.1 ml of diluted Folin–Ciocalteau reagent was added and the test samples were incubated at 30 °C for 30 min with resultant absorbance being measured at 750 nm.

gene sequences were constructed using the neighbor-joining method in MEGA software. 2.6. Analytical methods The dry weights of granules, the cell biomass, volatile suspended solids (VSS), and total suspended solids (TSS) were measured according to Standard Methods (APHA, 1998). The size of sludge granules was determined using a laser particle-size analysis system (Mastersizer Series 2600; Malvern, UK), or using an image analysis system. Washed granules and isolated pure culture were prepared for scanning electron microscopy (SEM) (S-2400, Hitachi Ltd., Tokyo, Japan) with the Ronatec system for energy dispersive spectroscopy (EDS) microanalysis. The SEM observations were conducted following fixing with 2.5% glutaraldehyde for 2 h, and dehydration via successive passages through 30%, 50%, 75%, 85%, 90%, 95% and 100% ethanol, followed by critical drying in a critical point dryer (HCP-2) (Hitachi Ltd., Tokyo, Japan). The supernatant was separated from 10 ml of original sludge/ granules using 5000g centrifugation for 15 min at 4 °C. The PBS buffer (pH 7.4) was added to the collected supernatant to its original volume of 10 ml. The supernatant was filtered using a 0.45-lm filter, and its polysaccharide (PS) and protein (PN) contents were determined; these contents were loosely bound extracellular polymeric substances (LB-EPS). The PBS buffer (pH 7.4) was added to the sediment to a final volume of 10 ml, and then subjected to ultrasonication at 40% amplitude with 2 s pulse for 10 min in an ice bath. The ultrasonicated suspension was centrifuged at 20,000g for 15 min at 4 °C and the supernatant was made up to 10 ml with PBS buffer. The supernatant was added with PBS buffer to a final volume of 10 ml. The PS and PN contents were determined as tightly bound EPS (TB-EPS). The collected sediment was re-dissolved in PBS buffer (pH 7.4) to a total volume of 10 ml; then 3 ml of 1 M NaOH solution was added. The suspension was stored for 1 h and was then centrifuged at 20,000g for 15 min at 4 °C and the supernatant made up to 10 ml with PBS buffer to collect the supernatant. The supernatant was filtered through 0.45-lm filter and its PS and PN contents determined as residual EPS. The PS con-

3. Results 3.1. Reactor performance The seed granules II in continuous-flow reactor disintegrated within 3-d of operation. Serious biomass washout was thereby noted that induced subsequent reactor failure (data not shown). Restated, the seed granules II were not structurally stable in continuous-mode operation. No further tests were done for these granules in this study. The aerobic granules in both reactor A and reactor B, which were fed with seed granules I, stably existed over the 216-d test. The corresponding granules had COD removal rates of 83–84% (Table 1). At test end, the TSS of the reactor A (AG) was 3.2 g l1, while that of reactor B (AGMBR) was 6.5 g l1. The VSS/TSS ratios were 62–64%. Over the entire test the pH of suspensions in both reactors ranged 7.4–7.9.

3.2. Granule characteristics Fig. 1 presents photographs of seed granules and aerobic granules collected from reactor B operated for 90 d and 210 d. The seed granules were irregularly shaped grey pellets roughly 2–3 mm in size (Fig. 1a). The settling test yielded a mean effective density of

Table 1 Performances for the continuous-flow reactors with aerobic granules. Reactor

COD removal (%)

MLSS (g l1)

MLVSS (g l1)

A B

84.2 ± 6.3 83.1 ± 6.3

5.1 ± 2.3 10.2 ± 5.2

3.2 ± 1.1 6.5 ± 3.4

LB-EPS (mg l1)

TB-EPS (mg l1)

Residue-EPS (mg l1)

PS

PN

PS

PN

PS

PN

26.9 33.7

832 739

10.6 20.3

273 325

66.5 102

980 1285

Fig. 1. Appearance of aerobic granules. (a) Seed granules; (b) AGMBR granules (210 d) (scale bar in cm).

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Table 2 Characteristics of seed granules and AGMBR granules. Characteristics

Seed granules

AGMBR granules (210 days)

Mean Mean Mean Mean Mean

2.3 1.4 1.2 0.47 0.37

1.9 1.5 1.3 0.87 0.67

size (mm) settling velocity (cm s1) effective density (kg m3) TSS (mg per granule) VSS (mg per granule)

granules of 1.2 kg m3. The mean TSS and VSS for individual granules were 0.47 mg and 0.37 mg/granule, respectively (Table 2). After reactor B operated for 90 d, seed granules turned into brown–yellow pellets with a relatively more regular shape (Fig. 1b). The size of granules increased slightly. Settling test results indicate that the mean effective density of granules increased to 4.0 kg m3. The mean TSS and VSS for individual granules were 0.60 mg, and 0.27 mg/granule, respectively. In other words, granules gained weight by incorporating inorganic matter (Table 2). After reactor B operated for 210 d, the granules became white, and changed into large (>5 mm) and small (<1 mm) granules with a reduced mean granule size. The mean effective density of granules decreased to 1.3 kg m3. The mean TSS and VSS for individual granules were 0.87 mg, and 0.67 mg/granule, respectively. That is, the granules gained weight with cell growth and with the accumulation of secreted extracellular polymeric substances. The proteins were enriched in mature granules, reaching 2300 mg l1, roughly 36% of the VSS of the suspension (Table 1). Enriched protein contents improve granules stability (Adav et al., 2009). The quantities of PS in the LB-EPS, TB-EPS and residue fractions were 26.9–33.7, 10.6–20.3 and 66.5–102.0 mg l1, respectively. The corresponding quantities of PN in the LB-EPS, TB-EPS and residue fractions were 739–832, 273–325 and 980–1285 mg l1, respectively. Hence, the AG and AGMBR systems effectively removed high levels of COD, with the PN/PS ratio of granules at 10–30 with >60% VSS (Table 1). Therefore, this study successfully operated aerobic granular sludge reactor at continuous-flow mode without granular breakdown. 3.3. Microbial community in the granule The DGGE fingerprints and band intensities in reactors A and B indicate that the microbial communities were very stable, except for those on d 97 (Fig. 2). The Bray–Curties similarity indices for

97" 66" 10 S 34 66 53 34" 10" 53" 97 50

60

70

80

90

100

Similarity (%)

Fig. 2. An ethidium bromide-stained polyacrylamide denaturing gradient gel (35– 70%) with DGGE profiles of 16S rDNA gene fragments for samples from MBR operated in continuous feeding mode and dandrogram of DGGE profiles of the bacterial communities. The sampling days are labelled at the top of each lane. (S = seed, 10–97 samples from MBR without membrane; bold numbers 10–97: samples from MBR with membrane, in dandrogram marked ’.)

Table 3 Bray–Curtis similarity indices for the samples from MBR operated in continuous feeding mode (S = seed, 10–97 samples from reactor A; bold numbers 10’–97’: samples from reactor B).

10 34 53 66 97 100 340 530 660 970

S

10

34

53

66

97

100

340

530

660

100.0 100.0 92.0 94.7 55.2 91.9 91.9 89.5 78.0 61.1

100.0 92.0 94.7 55.2 91.9 91.9 89.5 78.0 61.1

92.0 94.7 55.2 91.9 91.9 89.5 78.0 61.1

91.6 52.8 88.7 88.7 86.5 75.6 59.0

59.3 91.4 91.4 88.9 76.9 64.7

61.5 61.5 66.7 60.0 72.0

100.0 97.1 84.2 66.7

97.1 84.2 66.7

87.2 70.6

81.1

10–66 d granules had microbial structure of >91.6% similarity with the seed sludge (Table 3). No significant difference in microbial community structure for both reactors existed. The dandrogram identified only three major clusters. The Shannon diversity index for seed granules in the reactor A was 3.00 and was decreased marginally to 2.89 on d 66. Species richness and Shannon diversity indices suggest stable microbial diversity (Table 3). The granules from the reactor B (AGMBR) were used for construction of clone library to analyze the microbial diversity (Fig. 3). The randomly selected 50 clones were analyzed. The accession numbers for the clones deposited in GenBank are as follows: YC1 (GU062419), YC2 (GU062420), YC3 (GU062421), YC4 (GU062422), YC5 (GU062423), YC6 (GU062424), YC7 (GU062425), YC8 (GU062426), YC9 (GU062427), YC10 (GU062428), YC11 (GU062429), YC12 (GU062430), YC13 (GU062431), YC14 (GU062432), YC15 (GU062433), YC16 (GU062434), YC17 (GU062435), YC18 (GU062436), YC19 (GU062437), YC20 (GU062438), YC21 (GU062439), YC25 (GU062440), YC26 (GU062441), YC28 (GU062442), YC29 (GU062443), YC30 (GU062444), YC31 (GU062445), YC32 (GU062446), YC33 (GU062447), YC34 (GU062448), YC35 (GU062449), YC36 (GU062450), YC39 (GU062451), YC40 (GU062452), YC41 (GU062453), YC43 (GU062454), YC45 (GU062455), YC46 (GU062456), YC47 (GU062457), YC48 (GU062458), YC49 (GU062459), YC50 (GU062460), YC51 (GU062461), YC52 (GU062462), YC53 (GU062463), YC55 (GU062464), YC56 (GU062465), and YC57 (GU062466). The granules from reactor B (the AGMBR) were used for construction of a clone library for assessing microbial diversity (Fig. 3). The compositions of bacterial 16S rRNA gene libraries show that carbon sequestration and mineralizing Bacteroidetes dominated. Of the 50 sequenced clones, 28 were Bacteroidetes (56%), 15 were Proteobacteria (30.0%), and 7 were Actinobacteria (14%). Most clone sequences were members of the denitrifying bacterial community (YC-2, YC-6 to YC-11, YC-17, YC-18, YC-20, YC-21, YC-25, YC-26, YC-28, YC-29, YC-31, YC-32, YC-34, YC-35, YC-37, YC-43,YC-44, YC-46 to YC-49, YC-52 and YC-53), likely due to the high concentration of ammonium sulphate in the reactor medium and a useful level (sufficiently high) of electron donors (acetate). Phylogenetic analysis identified clustering of clones YC55, YC-15 and YC-34 with the Corynebacterium glutamicum strain, whose genome had the ftn and dps genes associated with iron storage. Although clone YC-12, YC-57, YC-51 and YC-5 belongs to microbial strain Actinomycetales and Acinetobacter sp. that have potential to precipitate carbonate, however, calcium carbonate precipitate was not noted in the granules (Table 4). The 16S rRNA gene sequences belonging to the Alcaligenaceae family were Alcaligenes (50%), clustered with biphenyl mineralizing Alcaligenes faecalis with a 99% bootstrap value, while clones YC-2 and YC-53 clustered with Comamonas sp. with a 94% bootstrap value (Fig. 3). Of the 28 clones affiliated with Bacteroidetes, most clone sequences were

Y.-C. Juang et al. / Bioresource Technology 101 (2010) 8051–8057

95

56

88

100

99 68

8055

Clone YC-55 Corynebacterium glutamicum strain [EF528293] 92 Corynebacterium acetoacidophilum [X84240] 100 Clone YC-15 Clone YC-34 Clone YC-12 Actinomycetales bacterium Gsoil 972[AB245399] 99 98 Humicoccus flavidus strain DS-52 [DQ321750] Microbacterium trichotecenolyticum [Y17240] Clone YC-33 100 Microbacterium xylanilyticum strain [EU741115] Clone YC-1 Clone YC-30 Acinetobacter sp. PD12 [AY673994] 99 Clone YC-57 99 Clone YC-51 Clone YC-5 Clone YC-2 88 94 Clone YC-53 99 Comamonas sp. ST18 [ FJ982927] Comamonas badia [AB164432] Clone YC-44 Clone YC-49 56 Alcaligenes sp. PAMU-1.5 [AB118220] 99 Alcaligenes faecalis strain WM2072 [AY548384] Clone YC-28 Castellaniella defragrans strain [AF508101] 99 Denitrobacter sp. BBTR53 [DQ337593] 100 Clone YC-32 81 Clone YC-17 89 Denitrobacter sp. CHNCT17 [ EF471227 ]

Clone YC-50

99

83

76

99

54 84

Petrimonas sulfuriphila strain BN3 [AY570690] Clone YC-45 Uncultured compost bacterium clone 2B[DQ346496] 79 Empedobacter sp. B202 [GQ232741] 86 Clone YC-48 Clone YC-36 Clone YC-56 Clone YC-46 Clone YC-29 Clone YC-21 93 Clone YC-39 Clone YC-7 Clone YC-26 Clone YC-37 Uncultured bacterium clone LR A2-8 [DQ988289] 99 Clone YC-31 Uncultured Bacteroidetes bacterium [AJ318144] Clone YC-35 Uncultured bacterium clone BH B34 [EU366941] Flavobacterium sp. ANU301 [EF192137] Clone YC-20 Clone YC-25 100 100 Flavobacterium odoratum [D14019] Clone YC-19 Uncultured Flavobacteriales bacterium [EU403677] Environmental 16s rDNA sequence [CU466738] 100 76 Clone YC-18

0.05

Fig. 3. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences. The sequences from the present study and close relatives were aligned by multiple alignments CLUSTAL W. Bootstrap analyzes were conducted on 1000 samples and percentage greater than 50% are indicated at the nodes. Scale bar, 0.05 changes per nucleotide position.

members of the Flavobacteriaceae family (Adav et al., 2009), a denitrifying bacterial community Clones YC-25 and YC-19 clustered with Flavobacterium odoratum and had a 100% bootstrap value. Phylogenic analysis shows that the majority of clone sequences categorized as Bacteroidetes were uncultured Bacteroidetes bacterium (Fig. 3).

3.4. Distributions of Ca and Fe in granule Fig. 4 presents the element distributions by SEM/EDX on the surfaces and in the interior of granules collected at different times. Both the surface and interior of seed granules (d 0) were 54–55% carbon and 45% oxygen. The granule surface had no phosphate,

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Table 4 Saturation indices for the supersaturated species (marked in black) under different pH and different total carbonates, calculated by Visual MINTEQ ver. 2.61.

Species

2 500 mg l1 ½CO2 þ HCO 3 þ CO3  pH 6 pH 7 pH 8

pH 9

2 2000 mg l1 ½CO2 þ HCO 3 þ CO3  pH 6 pH 7 pH 8

Ca3(PO4)2 (am2) Ca3(PO4)2 (beta) Ca4H(PO4)33H2O Fe(OH)27Cl3 Ferrihydrite (ferric oxyhydroxide) Ferrihydrite (aged) Goethite (a-FeO(OH)) Hematite (Fe2O3) Hydroxyapatite (Ca5(PO4)3(OH)) K-Jarosite (KFe3(OH)6(SO4)2 Lepidocrocite (c-FeO(OH)) Maghemite (Fe2O3) Magnesioferrite (Magnesium iron oxide) Na-Jarosite (NaFe3(OH)6(SO4)2) Strengite (hydrated iron phosphate)

6.77 4.02 3.35 8.49 4.64 5.15 7.35 17.1 0.13 9.45 6.47 9.30 8.24 3.70 6.30

0.08 2.67 3.34 9.85 6.90 7.41 9.61 21.6 12.8 7.32 8.73 13.8 18.7 1.57 3.33

3.19 2.52 3.09 8.05 4.21 4.72 6.92 16.2 1.05 8.62 6.04 8.44 7.29 2.22 6.46

60

3.69 0.94 0.27 9.52 5.97 6.48 8.68 19.8 5.46 10.45 7.80 12.0 12.8 4.69 6.23

D0_Surface D210_Surface D0_Interior D210_Interior

50 40

wt. (%)

30 10 8 6

1.52 1.23 1.90 10.2 6.93 7.44 9.64 21.7 9.69 10.3 8.76 13.9 16.7 4.59 5.34

0.37 0.30 0.20 9.44 5.90 6.41 8.61 19.6 6.22 10.7 7.73 11.8 12.5 4.27 6.74

1.60 2.27 2.21 10.2 6.91 7.42 9.62 21.6 10.1 10.7 8.74 13.8 16.4 4.30 5.90

pH 9 2.49 3.16 2.74 9.81 6.88 7.39 9.58 21.6 12.3 7.64 8.70 13.8 18.3 1.24 3.89

amounts of calcium and iron precipitates in granule interiors (Fig. 4). The CLSM staining test also shows high concentrations of calcium near the granule surface (Supplementary Fig. S1). We hypothesize that calcium and/or iron precipitates in the granule interior substantially enhanced the structural stability of aerobic granules. During MINTEQ calculations (Table 4), the following compounds likely formed as precipitates: calcium salts Ca3(PO4)2, Ca4H(PO4)3, Ca5(PO4)3(OH); and iron salts ferrihydrite (ferric oxyhydroxide), goethite, hematite, Jarosite, lepidocrocite, maghemite, and strengite (hydrated iron phosphate). That is, the phosphates and hydroxides were the main species in the precipitate.

4 2

4.2. Possible correlation between microbial activity and mineralization

0 C

O

P

Element

Ca

Fe

Fig. 4. Elemental distributions on the surface and in the interior of granule by SEM/ EDX collected at different operating times.

0.7% calcium and <0.2% iron, while the granule interior contained roughly 2% phosphate, 1.2% calcium and 1% iron. After d 210 of operation, the surfaces of AGMBR granules were 56% carbon, 43% oxygen, <1% of calcium or iron, and no phosphate. Conversely, the granule interior was composed of 44% carbon, 34% oxygen, 2.3% phosphate, and approximately 10% calcium and 10% iron. That is, the granule interior was enriched with calcium and iron compounds. The Visual MINTEQ version 2.61 calculated the species distribution for the granular system. Input data were as follows (g l1): 1.0 (NH4)2SO4; 0.2 NaCl; 0.2 MgSO47H2O; 0.02 FeCl3; 0.01 CaCl22H2O; 1.65 K2HPO4; 1.35 KH2PO4 and 0.01–2.0 CO2. The CLSM images of AGMBR granules stained with SYTOX Blue (pink), SYTO 63 (red), and Calcium Green-1 (green) were shown in Supplementary Fig. S1. Briefly, DNA fragments were distributed throughout the entire granule interior. Calcium was distributed principally at the rim regime of the granule interior. 4. Discussion 4.1. Metal precipitate and granule stability This study reveals for the first time that aerobic granules can exist stably in continuous-flow reactors, with or without a membrane, with sustainable COD degradation efficiency and a stable microbial community. The SEM/EDS analysis detected large

The precipitation of metals via the biomineralization pathway has been discussed (Naka, 2007a, 2007b). Zamarreno et al. (2009) examined strains that can form carbonate crystals. The diverse members of Bacteroidetes, such as those identified in granules in this study (Section 3.3), sequestrated and mineralized carbon (Lydell et al., 2004; Lueders et al., 2006). The denitrifying bacteria in granules in this study (Fig. 3) produced CaCO3 crystals in liquid medium (Boquet et al., 1973). The 16S rRNA gene sequences of clones YC-57, YC-51 and YC-5 belonged to Acinetobacter sp., which is capable of precipitating calcium carbonate as calcite and aragonite (Ferrer et al., 1998). The Flavobacterium sp., closely related to clones YC-25, YC-19, and YC-20 can precipitate calcium carbonate as calcite (Ferrer et al., 1998). Several clones–YC-15, YC-55, and YC34—belonging to Corynebacterium glutamicum exhibited two genes involved in iron storage, i.e., the ftn gene encoding ferritin and the dps gene encoding a protein that catalyzes oxidation of ferrous iron to ferric ions, which can precipitate in an alkaline environment (Wennerhold and Bott, 2006). Although previous studies revealed that biomineration can be carried on by numerous strains, the calculation using Visual MINTEQ version 2.61 estimated the occurrence of phosphate salts of calcium, and phosphate and hydroxide salts of iron (Table 4). The iron phosphates and iron oxides tended to precipitate in an alka2 line solution with high alkalinity ½CO2 þ HCO 3 þ CO3  (Table 4). The denitrification reaction occurring inside the granule interior by enriched denitrifiers (Fig. 3) converted formed nitrate from fed ammonium–nitrogen, yielding alkalinity (to increase pH and content of dissolved inorganic carbon). The bioreaction favours calcium and iron precipitation inside granules, thereby enhancing granule structural stability. We proposed that the granule stability was enhanced by gradually precipitating calcium and iron ions in-

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side granules, a combined effect of denitrification reaction inside granules with enriched denitrifying bacteria and the high concentrations of dosed phosphates for buffering suspension pH. 5. Conclusions The aerobic granules with long term stability were cultivated and tested, which could retain structural stability and biological activity during 216-d test in continuous-flow reactors. The granules interior were enriched with denitrifier bacteria and with high concentrations of phosphate salts. Calcium and iron alts were precipitated in the granule interior where high alkalinity and high concentrations of phosphates were presented, as a result of denitrification reactions occurred inside the granules. Acknowledgement This research is currently supported by the National Science Council (Taiwan). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.05.078. References Adav, S.S., Chen, M.Y., Lee, D.J., Ren, N.Q., 2007a. Degradation of phenol by Acinetobactor strain isolated from aerobic granules. Chemosphere 67, 1566– 1572. Adav, S.S., Lee, D.J., 2008. Single-culture aerobic granules with Acinetobacter calcoaceticus. Appl. Microbiol. Biotechnol. 78, 551–557. Adav, S.S., Lee, D.J., Lai, J.Y., 2008a. Intergeneric coaggregation of strains isolated from phenol degrading aerobic granules. Appl. Microbiol. Biotechnol. 79, 657– 661. Adav, S.S., Lee, D.J., Lai, J.Y., 2009. Proteolytic activity in stored aerobic granular sludge and stability loss. Bioresour. Technol. 100, 68–73. Adav, S.S., Lee, D.J., Ren, N.Q., 2007b. Biodegradation of pyridine using aerobic granules in the presence of phenol. Water Res. 41, 2903–2910. Adav, S.S., Lee, D.J., Show, K.Y., Tay, J.H., 2008b. Aerobic granular sludge: recent advances. Biotechnol. Adv. 26, 411–423. HA, A.P., 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC. Beun, J.J., Hendriks, A., van Loosdrecht, M.C.M., Morgenroth, E., Wilderer, P.A., Heijnen, J.J., 1999. Aerobic granulation in a sequencing batch reactor. Water Res. 33, 2283–2290. Chen, M.Y., Lee, D.J., Yang, Z., Peng, X.F., Lai, J.Y., 2006. Fluorescent staining for study of extracellular polymeric substances in membrane biofouling layers. Environ. Sci. Technol. 40, 6642–6646. Chen, Y.C., Lin, C.J., Chen, H.L., Fu, S.Y., Zhan, H.Y., 2009. Cultivation of biogranules in a continuous flow reactor at low dissolved oxygen. Water Air Soil Pollut. Focus 9, 213–221. Ferrer, R., Sarmiento, J.Q., Rivadeneyra, A., Bejar, V., Delgado, R., RamosCormenzana, A., 1998. Calcium carbonate precipitation by two groups of

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