Bioresource Technology 216 (2016) 562–570

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Bioresource Technology

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Optimizing granules size distribution for aerobic granularsludge stability: Effect of a novel funnel-shaped internalson hydraulic shear stress

http://dx.doi.org/10.1016/j.biortech.2016.05.0790960-8524/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Environmental Engineering, ZhejiangUniversity, No. 866 Yuhangtang Road, Hangzhou 310058, China.

E-mail address: lilithzhuliang@163.com (L. Zhu).

Jia-heng Zhou a,b, Zhi-ming Zhang a, Hang Zhao a, Hai-tian Yu a, Pedro J.J. Alvarez d, Xiang-yang Xu a,c,Liang Zhu a,⇑aDepartment of Environmental Engineering, Zhejiang University, Hangzhou 310058, ChinabCollege of Civil Engineering and Architecture, Zhejiang University of Technology, Hangzhou 310014, Chinac Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058, ChinadDepartment of Civil and Environmental Engineering, Rice University, Houston, TX 77005, USA

h i g h l i g h t s

� Novel internals selectively assignedhydrodynamic shear stress ongranules.

� More granules in novel reactor weresituated in an optimal size range.

� High pollutant removal performanceand stability were achieved in novelreactor.

� Existence of hydroxyapatite innercore further enhanced the stability ofgranules.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 February 2016Received in revised form 19 May 2016Accepted 20 May 2016Available online 24 May 2016

Keywords:Aerobic granular sludgeFunnel-shaped internalsHydraulic shear stressGranules size optimizationInorganic inner core

a b s t r a c t

A novel funnel-shaped internals was proposed to enhance the stability and pollutant removal perfor-mance of an aerobic granular process by optimizing granule size distribution. Results showed up to68.3 ± 1.4% of granules in novel reactor (R1) were situated in optimal size range (700–1900 lm)compared to less than 29.7 ± 1.1% in conventional reactor (R2), and overgrowth of large granules waseffectively suppressed without requiring additional energy. Consequently, higher total nitrogen (TN)removal (81.6 ± 2.1%) achieved in R1 than in R2 (48.1 ± 2.7%). Hydraulic analysis revealed the existenceof selectively assigning hydraulic pressure in R1. The total shear rate (stotal) on large granules was3.07 ± 0.14 times higher than that of R2, while stotal of small granules in R1 was 70.7 ± 4.6% in R2.Furthermore, large granules in R1 with intact extracellular polymeric substances (EPS) outer layerstructure entrapped hydroxyapatite at center, which formed a core structure and further enhanced thestability of aerobic granules.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction

The aerobic granular sludge process has become an emergingtechnology for domestic and industrial wastewater treatment inrecent decades (de Kreuk et al., 2007; Lee et al., 2010; vanLoosdrecht and Brdjanovic, 2014). Compared with activated

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sludge, granular sludge has multiple advantages such as densestructure, high biomass retention, and high pollutant removal effi-ciency (de Kreuk et al., 2005; Lee et al., 2010). These characteristicstend to enhance the separation process of sludge, which allows aclarifier to be combined with a bioreactor and reduces its footprintand energy cost by up to 75% and 25%, respectively (van Loosdrechtand Brdjanovic, 2014). However, the loss of granule stability fre-quently occurs during long-term operation, which limits the appli-cation of this method (Li et al., 2006; Li and Li, 2009; Sheng et al.,2010; Zhu et al., 2013).

Aerobic granules with large size tend to hinder the utilization ofnutrients and oxygen in granule interior because of the mass trans-fer limitations. Oxygen concentration profiles in aerobic granuleshave shown that oxygen was consumed at the surface layers witha thickness of 100–700 lm (Chiu et al., 2006; de Kreuk et al., 2007;Liu et al., 2010). The kinetic model simulation revealed themaximum nutrients penetration depth in acetate- and glucose-fed reactors was 1750 and 1250 lm, respectively (Chou et al.,2011). Limited oxygen concentrations in granule interior favorthe formation of an inner core with anaerobic microbes (Zhenget al., 2006). These anaerobic microbes decrease the pH of the innercore by producing acidic chemicals, which is proven to damage theouter shell and backbone matrix of aerobic granules (Adav et al.,2008, 2010). Moreover, nutrient limitations in large granules havealso been found to decrease the metabolic activity of functionalmicrobes, causing cavity structures in aerobic granules (Liu andTay, 2004). The cavities weaken the structural stability of granules,which subsequently cause granules disintegration under hydrody-namic shear stresses (Lee et al., 2010; Liu et al., 2010). However,simultaneous nitrification and denitrification (SND) during aera-tion in granules need certain thickness. Pervious study suggestedthat a maximal N removal was observed in granules with averagediameters of 1300 lm (de Kreuk et al., 2005). Considering bothsludge stability and nitrogen removal efficiency, an optimal gran-ule size of 700–1900 lm was proposed (Chen et al., 2011; Chouet al., 2011; Huang et al., 2011). Nevertheless, the mean size ofthe aerobic granules between 200 and 7000 lm was reported inprevious studies (Liu and Tay, 2004; Lee et al., 2010), and the per-formance and stability of the aerobic granular sludge process dif-fered with granule size distributions. Therefore, optimizinggranule size distribution for the structural stability and the perfor-mance of aerobic granules represents an important challenge.

To date, several strategies have been proposed to enhance thestability of the aerobic granular sludge process by suppressingthe overgrowth of large granules. Removal of large granules duringsettling (Li et al., 2006; Zhu et al., 2013) and selectively dischargeof large granules during aeration phases have been used (Li andLi, 2009; Sheng et al., 2010). However, selectively removing largegranules in a bioreactor is typically a complex process in realindustrial applications and can reduce the competitive advantageof granules compared to flocculent biomass, resulting in loosegranular structures (Li et al., 2006).

Increasing hydrodynamic shear stress has been proposed tosuppress the overgrowth of large granules (Adav et al., 2008; Liet al., 2011; Verawaty et al., 2013). A high hydrodynamic shearstress increases the attrition of granules, suppressing the over-growth of large granules, whereas a relatively low hydrodynamicshear stress favors the growth of granules size. However, a highhydrodynamic shear stress typically requires substantial amountsof energy, which increases the cost of industrial applications(Lochmatter et al., 2013).

Recently, new internals, such as baffles, stirring paddle andtubes, were developed to increase hydrodynamic shear stress inreactors (Baten et al., 2003; Soos et al., 2008). Compared with con-ventional reactors, airlift reactor (ALF) has a relatively constantshear stress (Baten et al., 2003). A stronger hydraulic shear stress

which favored the structural stability of granular sludge wasreported in stirring tank and ALF (Soos et al., 2008). Previous stud-ies showed sludge aggregate structure depends on the given stir-ring speeds (Soos et al., 2008). However, these internals led tohigher flow resistance and was difficult to construct. Moreover, ahigh hydrodynamic shear stress shifts granule size distributiontowards smaller sizes, which leads to an unfavorable size distribu-tion for SND process (Chen et al., 2011; Gonzalez-Gil and Holliger,2014). Therefore, a valid strategy for enhancing the stability andthe performance of aerobic granular sludge process via optimizinggranule size distribution is worthy of study.

In this study, a novel funnel-shaped internals was proposed forthe stable operation of an aerobic granular sludge process under alow superficial upflow air velocity. The hydraulic parameters ofaerobic granules were investigated using high-speed Charge-Coupled Device (CCD) visualization technology. The objectives ofthis study were to i) enhance the stability and pollutant removalperformance of an aerobic granular process by optimizing granulesize distribution, ii) characterize the effects of funnel-shaped inter-nals on hydraulics parameters of aerobic granules, and iii) advancemechanistic understanding of how hydraulic parameters optimizegranules size distribution.

2. Materials and methods

2.1. Experimental setup and operation

Two parallel Plexiglas sequencing batch bioreactors (SBRs) witheffective volumes of 10 L were used in this study. The reactors hadinternal diameters of 12.0 cm and height-to-diameter (H/D) ratiosof 10:1. The SBR cycle length was 4 h with 10 min of feeding,210 min of aeration, 10 min of settling and 10 min of withdrawal.The effluent was set at the height of 5 L volume in all the reactors,and the volumetric exchange ratio was 50%. Air was introduced bya fine-bubble porous aerator placed at the bottom of the reactors.The superficial upflow air velocity was kept at 0.65 cm s�1, whichwas much lower than the 2 cm s�1 in a conventional operation(Chen et al., 2007; de Kreuk et al., 2010; Wan et al., 2011). ForR1, a funnel-shaped steel-wire screen with a mesh size of2.5 mm was installed at a height of 70 cm. The steel-wire screenhad an inside diameter of 5 cm, which allow the upstream flowand granules pass through. There is a 1.0 cm gap betweenfunnel-shaped internals and sidewall of the reactor, which reducedflow resistance in downstream area. Steel-wire screen had a slopeangle of 60�, thus the granules did not stick to the screen duringsettling.

The kinetic model simulation revealed that maximum nutrientspenetration depth in granules was around 1250 lm, suggesting themaximum particle size should be smaller than 2.5 mm (Chou et al.,2011). Thus, in this study, a novel funnel-shaped internals withmesh size of 2.5 mm was proposed, which attempt to restrict thesize of large granules smaller than 2.5 mm. (Fig. 1).

Reactors were operated under an organic loading rates (OLR) ofabout 3 kg CODm�3 d�1. The influent’s COD concentration was1014 ± 27 mg L�1, and NH4

+-N, total nitrogen (TN) and total phos-phorus (TP) values were 45.7 ± 2.3 mg L�1, 89.3 ± 4.7 mg L�1, and15.13 ± 1.8 mg L�1, respectively. The composition of the syntheticwastewater used in this study was as follows (mg L�1): sodiumacetate, 1256; sucrose, 92.6; NH4Cl, 180.27; KH2PO4, 54.34;K2HPO4, 69.44; yeast, 250; peptone, 375; CaCl2, 80; MgSO4, 30. Inaddition, the synthetic wastewater contained a trace element solu-tion composed of the following components (mg L�1): H3BO3, 0.05;CuSO4�5H2O, 0.05; ZnSO4�7H2O, 0.05; AlCl3, 0.09; CoCl2, 0.05;MnSO4�H2O, 0.05; (NH4)6Mo7O24, 0.05; NiCl2�6H2O, 0.09; andFeSO4�7H2O, 0.05. The temperature was maintained at 25 ± 2 �C.

Fig. 1. Schematic of internals in the novel bioreactor (R1).

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The seed sludge was collected from an aerobic tank at the QigeMunicipal Wastewater Treatment Plant in Hangzhou, China. Thesludge volume index (SVI30) of the seed sludge was140 ± 17 mL L�1, and the biomass concentration was about4000 mg L�1. The seed sludge had loose morphology with meandiameter of 70.6 ± 3.6 lm.

2.2. Analysis of the hydraulic parameters in the bioreactors

An identical Plexiglas cylindrical tube was built to investigatethe velocity field of the granules. To avoid light refraction effectsinside the cylinder tube, the device was immersed in a rectangularPlexiglas tank filled with water. A light emitting diode (LED) lampwas situated in front of plexiglas tank as supplementary lighting. Ahigh-speed CCD camera (Vieworks, VH-2MG2-M42A0, Korea) with42 frames per second (42 fps) was installed on a lifting platformthat was horizontally controlled by a stepper motor in front ofthe Plexiglas tank. Images of the granules (at 1600 � 1200 pixelresolution) were taken using a TV lens (PENTAX, Japan). Theimages from the CCD camera were transferred through a GigE net-work and recorded on a computer (Zhou et al., 2015).

The background was divided into a dynamic threshold moduleby image processing. Different locations of granules in intervalimages (Dr) and interval time (Dt) permitted velocity calculations(t) (Díez et al., 2007; Zima et al., 2008). The velocity was calculatedas follows:

m ¼ DrDt

ð1Þ

where t is the velocity of granules (cm s�1), Dr is the positiondifference of granules in interval images (cm), and Dt is the timeinterval (s).

Each velocity field consisted of 2232 velocity vectors (24 vectorsalong axial direction � 93 vectors along vertical direction). Thegranule shear rate field was then calculated based on the granulevelocity field (Zima et al., 2008). The results of the shear ratesare presented in dimensionless form as follows:

sg ¼ jsg jjsmaxj ð2Þ

where sg is the dimensionless granules shear rates, sg is the shearrate (s�1), and smax is the maximum shear rate of two reactors (s�1).

The solid holdup (eg) distribution of the granules was calculatedafter removing background-related signals. The results of the gran-ule holdup are presented in the same dimensionless form as theshear rates.

The total shear rate (stotal) combined of the shear rate field andthe granule holdup distribution field was used to characterize theoverall hydraulic shear stress on the granules. The total shear ratewas calculated as follows:

stotal ¼X24;93

i¼1;j¼1

sgi;j � egi;j ð3Þ

where stotal is the total shear rate (s�1), sg is the shear rate (s�1), andeg is the granule holdup (%).

2.3. Confocal laser scanning microscopy (CLSM) observations of thegranules

Fluorescent staining and CLSM techniques were used to investi-gate the distribution of microbial cell, b-polysaccharide and deadcell in the granules. Granules were first fixed with 4%paraformaldehyde in phosphate-buffered saline (PBS, 0.01 mol L�1,pH 7.4 ± 0.2). Then, the granules were stained using fluorescentdyes of Calcofluor White (300 mg L�1, in normal saline (NS)) fromSigma (F3543-1G), Sytox Blue (20 lM, in NS) from Life Technolo-gies (S11348) and Syto 63 (2.5 lM, in NS) from Life Technologies(S11345). The stained samples were washed twice with NS toremove excess stain, stored at 4 �C (Chen et al., 2007) and thenexamined using a CLSM microscope (ZEISS, LSM710 NLO,Germany).

2.4. Vibratome observations of the granules

Vibratome technology combined with an optical microscopewas applied to observe the realistic interior geometries of the gran-ules. Granules were washed three times with PBS and stored over-night at 4 �C. Agarose gel with a concentration of 5% was heated toabove 90 �C in water and cooled to 40 �C. The granules were thenembedded in the agarose gel and cooled to room temperature.Granule sections of a certain thickness were obtained using avibratome (LEICA, VT 1000 S, Japan) and placed on microscopeslides. The morphology of the granule sections was observed usingan optical microscope (Nikon, Eclipse 80i, Japan) equipped with adigital camera (Nikon, Digital Sight DS-U3, Japan).

2.5. Three-dimensional (3D) reconstruction of the geometries of thegranules

3D reconstruction models were applied to demonstrate therealistic internal geometries of the granules. For the 3D reconstruc-tion, the morphologies of the granule sections with a thickness of30 lm were first obtained using the vibratome and optical micro-scope system. The background of the granule section images were

J.-h. Zhou et al. / Bioresource Technology 216 (2016) 562–570 565

then manually removed using image-processing software. Morethan 60 consecutive thin sections with thicknesses of 30 lm werestacked and reconstructed in 3D (3DS Max 2013).

2.6. Scanning electron microscopy (SEM) combined with energydispersive spectroscopy (EDS) and X-ray diffraction (XRD)

The external morphology of the granules along with the ele-mental distribution within the granules was examined by SEM+ EDS and XRD. SEM was applied to analyze the external structureof the sludge samples at different stages. The samples were firstfixed with 2.5% glutaraldehyde in a phosphate buffer (pH 7.0) formore than 4 h, washed three times in the phosphate buffer, thenpostfixed with 1% OsO4 in the phosphate buffer for 1 h beforewashing three times again in the phosphate buffer. For dehydra-tion, the sludge samples were first dehydrated by a graded seriesof ethanol (50%, 70%, 80%, 95%, and 100%) for approximately 15–20 min at each step; the samples were then transferred into a mix-ture of alcohol and iso-amyl acetate (v:v = 1:1) for approximately30 min and then into pure iso-amyl acetate for approximately1 h. Finally, the sludge samples were dehydrated in a critical pointdryer with liquid CO2 (Hitachi, Model HCP-2, Japan). The dehy-drated sludge samples were coated with gold-palladium andobserved by SEM (Hitachi, Model TM-1000, Japan) and SEM + EDS(Hitachi, S-3700N, Japan).

The sludge samples used for XRD analysis were first washedthree times with deionized water and then centrifuged to removesupernatants at 2000 rpm for 5 min (Allegra, 64R Centrifuge, USA).The deposits were calcined in an oven at 500 �C for 2 h to removeorganic fraction. XRD analysis was performed with a diffractome-ter (Shimadzu, XRD-6000, Japan) using 2h angles from 10� to 70�(Angela et al., 2011).

2.7. Other analytical methods

The chemical oxygen demand (CODCr), ammonia nitrogen con-centration (NH4

+-N), total nitrogen concentration (TN), mixedliquor suspended solids content (MLSS) and sludge volume index(SVI) were measured based on APHA standard methods (APAH,1998). The mean size and size distribution of the aerobic granuleswere measured by a laser particle size analysis system (QICPIC,Sympatec, Germany).

The dimensionless span value was then used to characterizegranules size distribution. The span value was calculated asfollows:

span ¼ ðd90 � d10Þd50

ð4Þ

where d10 is the diameter of granules larger than 10% of total parti-cle volume (lm), d50 is the mean diameter of granules (lm), and d90is the diameter of granules larger than 90% of total particle volume(lm).

Statistical analysis between all the variables was conducted byt-test using SPSS (SPSS 17.0). p < 0.05 was considered to be statis-tically different, and p < 0.01 was considered to be significantlydifferent.

3. Results

3.1. Performance of the novel funnel-shaped internals

After 10 days of operation, the SVI rapidly decreased from140 mL g�1 to approximately 40 mL g�1 in both reactors. However,the SVI in the novel bioreactor (R1) stabilized at approximately40 mL g�1 during the operation, whereas in conventional reactor

(R2) rapidly increased to 99 mL g�1 after 47 days due to granuledisintegration; the SVI in R2 then increased to 191 mL g�1 after87 days and failed to recover. The MLSS in R1 reached 6530 mg L�1

after granulation, and then stabilized above 8000 mg L�1; however,in R2, the MLSS reached 6300 mg L�1 after granulation, thendecreased rapidly to 1500 mg L�1 due to significant sludge washout (Supplementary Materials).

After aerobic sludge granulation, high removal efficiencies ofCOD and NH4

+-N were achieved in both reactors. The COD removalefficiency was 95.8 ± 3.7% for R1, 90.4 ± 2.4% for R2, and NH4

+-Nremoval efficiency was 98.1 ± 0.9% for R1, 88.1 ± 2.5% of R2, respec-tively. Removal efficiencies of COD and NH4

+-N in R1 was higherthan R2 (p < 0.01). Meanwhile, notable differences were observedin the TN removal efficiencies of two reactors. In R1, the TNremoval efficiency increased gradually, and finally stabilized at81.6 ± 2.1%, which was much higher than 48.1 ± 2.7% of R2 beforegranule disintegration (p < 0.01). Thereafter, granule disintegrationoccurred in R2, and removal efficiency dropped significantly (Sup-plementary Materials).

3.2. Size development and distribution of granules

Measurement of the particle size distribution and macroscopicobservation were used to investigate the development of granulesin both reactors. After 14 days of operation, the mean diameters ofboth reactors reached 500 lm, which indicated full granulation(Verawaty et al., 2013). The d90 diameter (the diameter of granuleslarger than 90% of total particle volume) of granules in R1increased slowly and stabilized at approximately 1887 ± 57 lm,whereas the mean diameter of granules reached 1743 ± 22 lm(Fig. 2a). By contrast, the granules size distribution fluctuated inR2. The d90 diameter of granules in R2 increased rapidly to4236 ± 37 lm by Day 40, which is much higher than R1, suggestingthat overgrowth of large granules had occurred (p < 0.01). Next,disintegration of the granules occurred, and the mean diametersignificantly decreased from 715 ± 24 lm to 253 ± 20 lm (Fig. 2b).The reactor then deteriorated after 87 days due to significantsludge washout. The macroscopic observations of the aerobic gran-ules showed that the particles in R1 had a relatively uniform sizedistribution, whereas those in R2 had a considerably uneven sizedistribution and an overgrowth of large granules before granulesdisintegration (Supplementary Materials).

A large span value indicates a wide size distribution, suggestinga significant size difference between the large and small granule,whereas a low span value indicates that the granules size distribu-tion is concentrated in a smaller range. The span value in R1 chan-ged marginally and stabilized at 0.91 ± 0.03 during operation,which suggested that R1 had a relatively uniform size distribution.However, the span value in R2 rapidly increased from 1.15 to 5.77after 40 days, indicating a significant broad size distribution(p < 0.01) (Fig. 2c). The optimal size range of the granules (700–1900 lm) has been proposed to favor stable operation and nutrientremoval in previous studies (Chen et al., 2011; Chou et al., 2011;Huang et al., 2011). Results showed that with a relatively smallmean diameter (715 ± 24 lm) and higher span value (5.77), fewergranules in R2 were situated in the optimal size range after granu-lation (less than 29.7 ± 1.1%). In contrast, up to 68.3 ± 1.4% of thegranules in R1 were situated in the optimal size range.

3.3. Structural characteristics of granular sludge

SEM images show that the large granules (with diameters above2.5 mm) in R1 had a relatively regular and dense structurecompared with those in R2 on Day 37. The granules in R1 consistedof dense bacterial populations with rod-shaped bacteria. A fewbacterial filaments were found near the granule surfaces.

Table 1Comparison of elements content in core and outer layer section of granules in R1.

Element Weight percent (%) Atomic percent (%)

Core Outer layer Core Outer layer

C 10.31 57.75 17.47 66.52O 45.61 34.61 58.02 29.93Na 0.23 2.98 0.20 1.80Mg 0.55 – 0.46 –P 13.49 1.87 8.86 0.83S 0.26 1.22 0.17 0.53Cl 0.18 – 0.10 –Ca 28.34 0.41 14.39 0.14Zn 1.02 – 0.32 –

Fig. 2. Variation of granule size distribution in R1 (a), Size distribution in R2 (b), and Comparison of span values (c).

566 J.-h. Zhou et al. / Bioresource Technology 216 (2016) 562–570

Conversely, the granules in R2 had an irregular appearance aftergranulation and were full of ravines and peaks (SupplementaryMaterials).

CLSM was used to investigate the distribution of microbial cells,b-polysaccharides and dead cells in granules. Result showed thatinterior without fluorescent intensity data were present in largegranules of both reactors. The b-polysaccharides (blue-stained),which are believed to act as the EPS backbone matrix in granularsludge, formed the dense outer layer of granular sludge in R1,whereas nucleic acids from live cells (red-stained) accumulatedclose to b-polysaccharides. Dead cells were present near thecore of the granule but more closely than b-polysaccharides. Itappears that the large granules in R1 had intact outer-layerb-polysaccharides structures, whereas those in R2 showed incom-plete outer-layer b-polysaccharides structures (SupplementaryMaterials).

Because the SEM and CLSM images were not able to observe theinternal structures of the large granules, vibratome technologycombined with microscopy was used to investigate the realisticinternal geometries of the granules. Ten large granules (withdiameters above 2.5 mm) from each reactor after 37 days ofoperation were cut across their centers using the vibratome for fur-ther examination. The images of the large granules sections fromR1 showed dense core structures and sharp demarcations betweenthe core and outer layer, whereas a hollow inner structure and con-nected irrigated channels were observed in large granules from R2(Supplementary Materials).

The granules in R1 were then analyzed by SEM + EDS and XRD.The large granules from R1 were cut across for SEM + EDSexamination. Results showed that the large granules in R1 werecomposed of a sharp demarcation core (crystal-like) and an outerlayer (bacteria-like) (Supplementary Materials). The EDS resultsrevealed the core sections of granules in R1 had much higherconcentrations of Ca and P (28.34% and 13.49% weight percent,respectively) and relatively low C concentration (10.31% weightpercent) compared to the outer layer (0.41%, 0.87% and 57.75%weight percent, respectively) (Table 1). The sludge samples fromR1 were then subjected to XRD analysis. The diffractogram resultsof large granules in R1 showed that most of the stronger peaks

J.-h. Zhou et al. / Bioresource Technology 216 (2016) 562–570 567

coincide with those of hydroxyapatite (Ca5(PO4)3OH) patternsafter being compared with the standard card (SupplementaryMaterials).

3.4. 3D reconstruction of aerobic granule

3D reconstruction models were applied to reconstruct the real-istic internal geometry of large granules (with diameters above2.5 mm) in both reactors after 37 days of operation. The 3D recon-struction models consisted of more than 60 layers of thin sectionswith thicknesses of 30 lm. As shown in the 3D models, granules inR1 had a distinctive inner core, and a few dead-end cavitieswrapped by an intact outer layer structure. In contrast, granulesin R2 exhibited a connected irrigated channel structure with evi-dent pores and a connected outer layer structure (SupplementaryMaterials).

Fig. 3. Hydraulic parameters analysis of large granules: (a) Velocity distribution in R1distribution in R2 (e) Holdup distribution in R1 (f) Holdup distribution in R2.

3.5. Hydraulic analysis of aerobic granular sludge process

The novel funnel-shaped internals was found to successfullyenhance the stability and pollutant removal performance of byoptimizing granule size distribution. To further discern the effectsof funnel-shaped internals on hydraulics parameters of aerobicgranules, velocity fields, shear rate fields and holdup distributionof granules were characterized. Because of the strong blockingeffect of bubbles on visualization, hydrodynamic parameters inthe center of reactor were not characterized (depicted in white).

Velocity fields showed that the large granules (with diametersabove 2.5 mm) in R2 circulated from the bottom to the top of reac-tor. The large granules in the bottom of R2 were first entrained bybubbles generated from the aerator, then moved to the top portionof reactor in the central upstream area. After reaching the top ofthe reactor, the large granules moved downward along the side-wall of the reactor to the bottom portion (Fig. 3b). The movement

(b) Velocity distribution in R2 (c) Shear rates distribution in R1 (d) Shear rates

Fig. 4. Comparison of total shear rate (stotal) on large granules in R1 andconventional reactor under different superficial upflow air velocity.

568 J.-h. Zhou et al. / Bioresource Technology 216 (2016) 562–570

of large granules in R1 showed distinctive differences from that ofthe R2. Most of the large granules in R1 first moved to the top por-tion in the central upstream area and were then intercepted by thefunnel-shaped internals screen when moving downward along thesidewall. The funnel-shaped internals performed as a deflector,which caused the large granules to move along it. The largegranules were then entrained by upstream bubbles andmoved intothe top portion of the reactor again. Results showed that the largegranules were intercepted by the funnel-shaped internals andcirculated at the top portion of R1 (Fig. 3a).

The shear rate fields of large granules (with diameters above2.5 mm) were then deduced from the granules’ velocity gradient.The results and descriptions of shear rates are presented in dimen-sionless forms. The shear rate of the large granules in R1 showedhigh stress areas (depicted in red) appeared in the top portion ofthe reactor bounded by the funnel-shaped internals (Fig. 3c). Incontrast, the shear rate of large granules in R2 was relatively lowerand uniform (Fig. 3d).

Due to the funnel-shaped internals, a significant high holdup oflarge granules (depicted in pink) in R1 appeared in its top portion,which was bounded by the funnel-shaped internals (Fig. 3e). Theresults showed that the holdup distribution of the large granulesin R2 was relatively uniform and marginally increased at the bot-tom portion of reactor due to gravity (Fig. 3f).

The overall hydraulic shear stress of granules was needed toevaluate the hydraulic effects of funnel-shaped internals withinreactors. However, the overall hydraulic shear stress was difficultto characterize due to the variations in shear rates and holdup dis-tributions at different positions within the reactors. In this study,the total shear rate (stotal) combined with shear rate fields andthe holdup distribution fields were used to characterize the overallhydraulic shear stress on granules. Results show that by using thefunnel-shaped internals, stotal of large granules (with diametersabove 2.5 mm) in R1 was 3.07 ± 0.14 times higher than that in R2under same superficial upflow air velocity.

The velocity field also showed that small granules (with diam-eters below 0.6 mm) in R1 could pass though the funnel-shapedinternals without hindrance (Supplementary Materials). The shearrate fields showed that the hydrodynamic shear rate on small gran-ules decreased in R1 (Supplementary Materials). Combined withshear rate fields and holdup distribution fields, stotal of small gran-ules (with diameters below 0.6 mm) in R1 was 70.7 ± 4.6% in R2(Supplementary Materials). stotal on the large granules in R1increased, whereas the value on small granules decreased provedthat the hydraulic pressure had been selectively assigned by usingthe funnel-shaped internals.

Moreover, the velocity, shear rate and holdup distribution fieldsof conventional reactor under different superficial upflow airvelocity were characterized, and the overall hydraulic shear stress(stotal) of large granules was deduced (Supplementary Materials).stotal on the large granules in conventional reactor showed thatwith the increase of upflow air velocity, stotal increased slightly,which suggested increasing aeration intensity is a less efficientway to suppress the overgrowth of large granules. However, byusing funnel-shaped internals in R1, stotal was even higher(2.07 ± 0.13 times) than conventional reactor with only 16.25% ofits aeration intensity (0.65 cm s�1 vs 4.0 cm s�1) (Fig. 4).

4. Discussion

4.1. Effect of novel funnel-shaped internals on granule stability

In this study, a significantly lower superficial upflow air velocityof 0.65 cm s�1 was applied in both reactors, which frequently resultedin the overgrowth of large granules and reactor deterioration

(Liu and Tay, 2002; Chen et al., 2007; de Kreuk et al., 2010;Wan et al., 2011). However, by using novel funnel-shaped internalsas hydrodynamic control strategy, the stability and pollutantremoval performance of an aerobic granular process wasenhanced. The total shear rate (stotal) on large granules in R1 selec-tively increased, which increased the attrition on large granules,while the stotal on small granules decreased, which facilitated thegrowth of the small granules. As a result, up to 68.3 ± 1.4% of thegranules in R1 were situated in the optimal size range(700–1900 lm) compared with less than 29.7 ± 1.1% in R2.

Pervious studies showed only 60% TN removal efficiency can beachieved with classical SND strategies (de Kreuk et al., 2005;Lochmatter et al., 2013). However, with optimized granule size dis-tribution, the TN removal efficiency in R1 finally stabilized at81.6 ± 2.1%. Considering mass transfer limitations, an optimalgranule size of 700–1900 lm was proposed for the best nitrogenremoval efficiency (Chen et al., 2011; Chou et al., 2011; Huanget al., 2011). By using funnel-shaped internals, more granules weresituated in the optimal size range (700–1900 lm), which enhancedpollutant removal performance in R1.

Previous studies showed that during conventional operation,biomass would eventually decay and produce empty pores in thelarge granules due to diffusion and biological activity (Tay et al.,2003). Low pH values occurred during biomass decay and hydro-lyzed the b-polysaccharides structures, which damaged the EPSmatrix and left connected channels in the granules (Adav et al.,2008, 2010). However, neither the pore structure nor channelstructure was found in large granules from R1. Results showed thatthe large granules in R1 had a dense and intact b-polysaccharidesouter layer structures, whereas those in R2 had a weak and incom-plete outer layer structures with connected irrigated channels. It ispostulated that selectively increasing the shear stress on largegranules in R1 not only stimulate the secretion of EPS (Liu andTay, 2002), but also increased attrition rate, which effectively cre-ated a dense EPS matrix to resist hydrolysis damage, and finallyformed an intact outer structure under low superficial upflow airvelocities (Adav et al., 2008).

Concurrently, 3D model reconstruction combined with EDS andXRD analyses further revealed the existence of the dense core ingranules in R1, whereas those in R2 showed pores in theirstructures. Results showed that the primary constituent of thecores in R1 was hydroxyapatite (Ca5(PO4)3OH), which was agreedwith previous studies (Angela et al., 2011; Li et al., 2014). Thedense core structure of the large granules in R1 may suppress

J.-h. Zhou et al. / Bioresource Technology 216 (2016) 562–570 569

the solubilization of the inner structure, which reinforces the sta-bility of the large granules in R1. In contrast, the connected irri-gated channels and pores structure in granules from R2 result inweak structures, and disintegrated before being washed out ofthe reactor. It is assumed that the hydroxyapatites formed in theouter layers (and the inner parts of R2) of the granules were diffi-cult to retain due to hydraulic disturbance, as they were easilywashed out into the bulk liquor through the channels in the gran-ules’ structures. However, with an intact EPS matrix in the outerlayer structure, the hydroxyapatite crystals in R1 may mitigatethe effects of hydraulic disturbances, keeping the particles tightlywrapped in EPS material and allowing a dense core structure todevelop.

4.2. Hydrodynamic control strategy in the novel bioreactor

In this study, a funnel-shaped internals was proposed to selec-tively assign hydraulic shear stress on granules of various sizes inR1 under low superficial upflow air velocity. This is to our knowl-edge the first demonstration that a hydrodynamic control strategycan optimize granule size distribution without additional energyconsumption.

The changes of granules’ velocity lead to tangential and normalaccelerations, resulting in the increase of hydrodynamic shearrates on granules (Díez et al., 2007; Zima et al., 2008). The largegranules in R2 moved in a steady flow, which suggested low radialand tangential accelerations. Based on these facts, relatively lowand uniform hydrodynamic shear rates occurred. However,funnel-shaped internals in R1 acted as a deflector, which forcedlarge granules to move along it. The large granules were thenentrained by upstream bubbles and moved circularly in the topportion. Consequently, a significant change of granules velocityoccurred, which led to high radial and tangential accelerations onthe large granules, significantly increasing the shear rate in thetop portion of R1. Concurrently, the granule holdup distributionin R2 was relatively uniform, while a high holdup of the large gran-ules appeared in the top portion of R1 because the large granuleswere intercepted by funnel-shaped internals. Combined with shearrates and granules holdup, the total shear rate (stotal) on large gran-ules in R1 significantly increased. Meanwhile, as the input energyinto the reactor was constant, the total shear rate on small granulesin R1 decreased due to the increasing hydrodynamics shear stresson large granules. Consequently, selectively assigning hydraulicshear stress on granules in R1 was achieved.

5. Conclusions

A novel funnel-shaped internals was proposed to optimizegranule size distribution by selectively assigning hydrodynamicshear stress on granules. By using the novel funnel-shaped inter-nals, total shear rate (stotal) on large granules in R1 was3.07 ± 0.14 times higher than R2, while stotal of small granules inR1 was 70.7 ± 4.6% in R2 without requiring additional energy.Overgrowth of large granules was effectively suppressed and opti-mal size distribution achieved in R1. The intact outer EPS layer ofgranules entrapped hydroxyapatite at center of granules, allowinga stable core structure to form, which further enhanced the stabil-ity of aerobic granules in R1.

Acknowledgements

This study was funded by the National Natural ScienceFoundation of China (No. 51478416), the Public Projects ofZhejiang Province (No. 2014C33017), the Natural ScienceFoundation of Zhejiang Province (No. LY13E080003), the National

Key Technologies Research and Development Program of China(No. 2013BAC16B04), and the Fundamental Research Funds forthe Central Universities.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2016.05.079.

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