Research review paper

Interactions

. . . .n, and. . . .. . . .

Biotechnology Advances 26 (2008) 411423

Contents lists available at ScienceDirect

Biotechnology Advances

j ourna l homepage: www.e lsev ie r.com/ locate /b iotechadv2.3. Chemical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4142.3.1. Biological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415

2.4. Granule applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4152.4.1. Treating toxic organic wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4152.4.2. Treating dairy wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4162.4.3. Removal of nitrogen and phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4162.4.4. Removal of heavy metals or dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4162.4.5. Removing particulate matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4162.4.6. Removal of nuclear waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416Abbreviations: AGSBR, aerobic granular sludge memmicroscope; COD, chemical oxygen demand; Con A, cuorescence in situ hybridization; FITC, uorescein-isotsuspended solids; MTBE, methyl t-butyl ether; NOB, nitscanning electron microscope; SBR, sequencing batch revolume index; UASB, upow anaerobic sludge blanket; Corresponding author. Department of Chemical Eng

E-mail address: djlee@ntu.edu.tw (D.-J. Lee).

0734-9750/$ see front matter 2008 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2008.05.002. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

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2.2. Granule characterization . . . .

2.2.1. Physical parameters . . .2.1.2. Feed composition2.1.3. SBR operation . .. . . . . . . . . . . . . . . . .

. . . .Contents

1. Introduction . . . . . . . . . .2. Granule formation, characterizatio

2.1. Granule formation . . .2.1.1. Seed sludge . . .industrial and municipal wastewaters. Factors affecting granulation, granule characterization, granulationhypotheses, effects of different operational parameters on aerobic granulation, response of aerobic granulesto different environmental conditions, their applications in bioremediations, and possible future trends weredelineated. The review attempts to shed light on the fundamental understanding in aerobic granulation bynewly employed confocal laser scanning microscopic techniques and microscopic observations of granules.

2008 Elsevier Inc. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413Extracellular polymeric substancesStructurerecent research development in aerobic biogranulation technology and applications in treating toxicKeywords:Aerobic granulesMechanisms

granulation and the strategyand environmental factors have not yet been clearly described. This paper provides an up-to-date review onAerobic granular sludge: Recent advances

Sunil S. Adav a, Duu-Jong Lee a,, Kuan-Yeow Show b, Joo-Hwa Tay c

a Department of Chemical Engineering, National Taiwan University, Taipei, Taiwanb University of Tunku Abdul Rahman, 13 Jalan 13/6, 46200 Petaling Jaya, Selangor Darul Ehsan, Malaysiac Institute of Environmental Science and Engineering, Nanyang Technological University, Innovation Centre, Block 2, Unit 237, 18 Nanyang Drive, 637723, Singapore

A B S T R A C TA R T I C L E I N F O

Article history:Received 21 February 2008Received in revised form 10 April 2008Accepted 6 May 2008Available online 19 May 2008

Aerobic granulation, a novel environmental biotechnological process, was increasingly drawing interest ofresearchers engaging in work in the area of biological wastewater treatment. Developed about one decadeago, it was exciting research work that explored beyond the limits of aerobic wastewater treatment such astreatment of high strength organic wastewaters, bioremediation of toxic aromatic pollutants includingphenol, toluene, pyridine and textile dyes, removal of nitrogen, phosphate, sulphate and nuclear waste andadsorption of heavy metals. Despite this intensive research the mechanisms responsible for aerobic

to expedite the formation of granular sludge, and effects of different operationalbrane bioreactor; AOB, ammonium oxidizing bacteria; AUFB, aerobic upow uidized bed; CLSM, confocal laser scanningoncanavalin A; DO, dissolved oxygen; EEM, excitationemission matrix; EPS, extracellular polymeric substances; FISH,hiocyanate; GAO, glycogen non-polyphosphate-accumulating organisms; MBR, membrane bioreactor; MLSS, mixed liquorrite oxidizing bacteria; OLR, organic loading rate; PAO, polyphosphate-accumulating organisms; PNP, p-nitrophenol; SEM,actor; SG, specic gravity; SOUR, specic oxygen utilization rate; SRT, solid retention time; SS, suspended solids; SVI, sludgeVSS, volatile suspended solids.ineering, National Taiwan University, Taipei, 10617, Taiwan. Tel.: +886 2 232532; fax: +886 2 23623040.

l rights reserved.

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421crobial species . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421

microbial aggregates and their densities were much higher than that

Recent development in aerobic biogranulation technology is nowreviewed and presented in this paper. Materials covered by existingreviews are not duplicated herein. Factors affecting granulation,granule characterization, granulation hypotheses, effects of differentoperational parameters on aerobic granulation, response of aerobicgranules to different environmental conditions, their applications in

412 S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423of conventional activated sludge. In addition, the aerobic granuleswere known to exhibit attributes of:

1) Regular, smooth and nearly round in shape2) Excellent settleability3) Dense and strong microbial structure4) High biomass retention5) Ability to withstand at high organic loading6) Tolerance to toxicity

Because of the unique granule attributes, the aerobic granula-tion technology was recently developed for treating high strengthwastewaters containing organics, nitrogen, phosphorus, toxic sub-stances and xenobiotics (Jiang et al., 2002; Moy et al., 2002; Tayet al., 2002b; Lin et al., 2003; Adav et al., 2007a,b,c,d; Adav and Lee,

Fig. 1. Appearance of granules cultivatedwith synthetic substrate containing 1000mg L1

acetate and with mineral concentrations that were half of those of Tay et al. (2001a,b).Flowing air for aeration air was supplied at a rate of 2 cm s1. The SBR was operated with4 min of inuent lling, 202 min of aeration, 30 min of settling and 4 min of efuent3. Granulation mechanism . . . . . . . . . . . . . . . . . . . . .3.1. Granulation steps . . . . . . . . . . . . . . . . . . . . .3.2. Selective pressure driven hypothesis . . . . . . . . . . . .3.3. EPS enhanced granulation . . . . . . . . . . . . . . . . .3.4. Granulation models . . . . . . . . . . . . . . . . . . . .

4. A closer look . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1. EPS and granule stability . . . . . . . . . . . . . . . . . .4.2. Intra-granular transport . . . . . . . . . . . . . . . . . .4.3. Strain interactions . . . . . . . . . . . . . . . . . . . . .

5. Summary and perspectives . . . . . . . . . . . . . . . . . . . .5.1. Thermophilic aerobic granules . . . . . . . . . . . . . . .5.2. Combined processes . . . . . . . . . . . . . . . . . . . .5.3. Cultivation of aerobic granules with genetically engineered mi

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Aerobic granules were considered to be a special case of biolmcomposing of self-immobilized cells. During the last 20 years,intensive research in the eld of biological wastewater treatmentand other applications demonstrated that biolms were often moreefcient for water purication than suspended activated sludge. Todate, the application of aerobic granular sludgewas regarded as one ofthe promising biotechnologies in wastewater treatment. The rstpatent was granted by Heijnen and van Loosdrecht (1998). de Kreuket al. (2007a,b) provided comments on the state of the art for theaerobic granulation process. Liu and Tay (2004) and Maximova andDahl (2006) provided an up to date summary of the current under-standing towards the bioaggregation processes.

Granular sludge was rst described for strictly anaerobic sys-tems in 1980 (Lettinga et al., 1980) and only by the late 1990s hadthe formation and application of aerobic granules been reported(Morgenroth et al., 1997, Beun et al., 1999, Dangcong et al., 1999).The anaerobic granulation technology exhibited several drawbacksthat included a long start-up period, a relatively high operatingtemperature, unsuitability for low strength organic wastewater, andlow efciency in the removal of nutrients (N and P) from waste-water. This resulted in the development of aerobic granular technol-ogy which became a popular topic of discussion for environmentalengineers.

Compact structured, biologically efcient aerobic sludge granuleswith wide diverse microbial species and excellent settling capabilitieshave been developed in sequencing batch reactors (SBR) (Morgenrothet al., 1997; Beun et al., 1999; Tay et al., 2001a; Yang et al., 2003; Liuand Tay, 2004, Adav et al., 2007a). Formation by self immobilization ofbacteria as hypothesized by several researchers (Kim et al., 2004;McSwain et al., 2004a; Qin et al., 2004a,b; Wang et al., 2004; Hu et al.,2005; Liu et al., 2005), the aerobic granules were densely packed2008a).bioremediations, and possible future trends are delineated. Applica-tions in treating municipal and toxic industrial wastewaters as wellas useful information on exploring the underlined mechanisms arealso highlighted. The review aims at providing understanding onaerobic granulation using newly employed confocal laser scanningmicroscope (CLSM) techniques and microscopic observations ofgranules.

2. Granule formation, characterization, and applications

Granulation was affected by a number of operational parameters,such as seed sludge, substrate composition, organic loading rate,feeding strategy, reactor design, settling time, exchange ratio, andaeration intensity (hydrodynamic shear force). It seems that therewasa relatively small operational window for the successful cultivationof aerobic granules. Under favorable conditions, the much desiredsuper granules could be formed. Fig. 1 shows the appearance of theacetate-fed granules cultivated using an SBR following 60 days ofincubation. A maximum granule size of 2.6 cm is seen. Some granulesexhibited a dark core, indicating the presence of an anaerobic zonewithin the aerobic granule.withdrawal. Incubation time=60 d. Maximum granule size=2.6 cm.

2.1. Granule formation

2.1.1. Seed sludgeSeed sludge, feed compositions and SBR operational parameters

(pH, temperature, cycling time, and others) affect granulation process(Liu and Tay, 2004). In most studies, aerobic granules were cultivat-ed with activated sludge seed. The bacterial community residing inactivated seed sludge was important for aerobic granulation processas the hydrophilic bacteria would be less likely to attach to sludgeocs compared with the hydrophobic counterpart, which constitutesthe majority of free bacteria in the efuent from full-scale treatmentplants (Zita and Hermansson, 1997). The greater the number ofhydrophobic bacteria in the seed sludge the faster the aerobicgranulation with excellent settleability (Wilen et al., 2007).

2.1.2. Feed compositionVarious substrates were used to cultivate aerobic granules, in-

cluding glucose, acetate, phenol, starch, ethanol, molasses, sucroseand other synthetic wastewater components (Liu and Tay, 2004; Tay

2.1.3. SBR operationIt appeared that aerobic granules were successfully cultivated only

in SBR. The cyclic operation of SBR consisted of inuent lling,aeration, settling and efuent removal. Liu and Tay (2007a) noted that,when cycle time increased from 1.5 to 8 h, the specic biomass growthrate of granular sludge decreased from 0.266 to 0.031 d1, while thecorresponding biomass growth yield (Yobs) decreased from 0.316 to0.063 g VSS g1 COD. Furthermore, the granules cultivated at 1.5 hcycle time were the biggest in size while the granules cultivated at4 h cycle time were the most compact ones compared with thosecultivated at other cycle times. Liu and Tay (2007b) noted the ex-istence of optimal starvation time for enhanced granule stability. Atan OLR of 6.0 kg COD m3 d1, the compact aerobic granules grew insize but gradually lost their stability corresponding with lamentousgrowth (Zheng et al., 2006).

The aeration period in SBR operation consisted of two phases: adegradation phase in which substrate depleted to a minimum, fol-lowed by an aerobic starvation phase inwhich substrate was no longeravailable. A long starvation period weakened the granule stability

ulein o

413S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423et al., 2002a; Tay et al., 2004a; Zheng et al., 2005, Adav et al., 2007a,b,c,d,e). Information on granule cultivationwith real wastewater was alsoreported (Arrojo et al., 2004; de Bruin et al., 2004; Schwarzenbecket al., 2005; Su and Yu, 2005,Wang et al., 2007a,b,c). The Cultivation ofgranules with phenol as a carbon and energy source demonstrateddominance of the Proteobacterium (Jiang et al., 2004b; Whiteley andBailey 2000; Adav et al., 2007c). Adav and Lee (2008b) cultivatedsingle strain granules from phenol-containing wastewaters.

The positive, divalent and trivalent ions, such as Ca2+, Mg2+, Fe2+

and Fe3+ could bind to negatively charged cells to form microbialnuclei (Mahoney et al., 1987). Jiang et al. (2003) revealed that theaddition of Ca2+ ions accelerated aerobic granulation. Granules wereformed in 16 days when 100 mg Ca2+ L1 was added in the feedwhereas 32 days were required without the addition of Ca2+.

The pH of medium signicantly affected the microbial growthrate. Oxidation at high OLR produced sufcient CO2 to reduce pHin unbuffered solutions (McSwain et al., 2004a). Fungi grew well atlow pH and may contribute signicantly to the initial granulation(McSwain et al., 2004a; Beun et al., 1999; Williams and de los Reyes,2006) since they could release protons in exchange for NH4+ in thesolution (Deacon, 2006), thereby further reducing the pH in thereactor. Yang et al. (2008) noted that, aerobic granulation at pH 4.0 inthe presence of a fungus produced a granule size of about 7mm, whileat pH 8.0 when the granulationwas controlled by bacteria the granulesize reached only 4.8 mm. Up to now, however, the effects of pH onthe inoculum selection and aerobic granulation have not been fullyaddressed.

Fig. 2. Effect of carbon source on the dominancy of the microbial strains. (a) acetate granwastewater containing 400mg L1 acetate and 400mg L1 phenol and operated with 5 m

1Air for aeration was supplied at a rate of 3 cm s .(Wang et al., 2005a, 2006). McSwain et al. (2004b) enhanced aerobicgranulation by intermittent feeding. Pulse feeding to the SBR con-tributed to compact aerobic granules. Although starvation was pro-posed not to be a prerequisite for aerobic granulation (Liu and Tay,2008; Liu et al., 2007), increases in hydrophobicity on carbon-starvation had been reported (Sanin et al., 2003; Sanin, 2003; Chenand Strevett, 2003). The impact of feast-famine operation ongranulation process was not well understood.

Short settling time caused washout of poorly settled suspendedbiomass and retained only well settled granules (Qin et al., 2004a,b).Studies have indicated that short settling time could enhance aerobicgranulation (Jiang et al., 2002; Lin et al., 2003; Liu et al., 2003a; Yanget al., 2003; McSwain et al., 2004a; Hu et al., 2005).

Adav et al. (2007b) compared the granulation processes in threeidentical reactors fed with phenol-containing wastewater and aer-ated at different intensities (13 L air min1). At low aeration intensity(1 L min1), no granules were formed. At high aeration rate (3 L min1),mature and stable granules (11.5 mm) with a compact interior wereformed. At intermediate intensity (2 Lmin1), large granules (33.5mm)with overgrown laments were formed (Fig. 2). While Liu and Liu(2006) accounted for the possible causes to lament overgrowth,Adav et al. (2007b) claimed that intermediate aeration could neitheryield sufcient oxygen supply nor break down overgrown laments,hence leading to SBR failure. Restated, strong shear force would notonly compact aggregates into granules, but also would provide suf-cient oxygen to suppress lament growth for a long-term operationalstability.

(b) phenol granule. Granules were cultivated in sequential batch reactor with syntheticf inuent lling, 220min of aeration,10min of settling and 5min of efuent withdrawal.

Keeping the operation at an appropriate solids retentions time(SRT), or the so-called sludge age, was essential to maintain granulestability (Li et al., 2007a). Li et al. (2007b) investigated aerobicgranulation in membrane bioreactor, and measured the total extra-cellular polymeric substances (EPS) contents on day 5 as 39.6mggSS1,and as 749.5 mg gSS1 on day 28. Nagaoka et al. (1996) had suggestedthat EPS adsorbed on the surface of aerobic biogranules led to in-creased water content and loose-structure of biogranules.

Most aerobic granular sludge research was carried out at roomtemperature (2025 C). The SBR operation at 8 C yielded granules,which were irregular in shape and with outgrowth of lamentousorganisms that caused severe biomass washout and unstable opera-tion (de Kreuk et al., 2005b). The corresponding denitrifying capacityand nutrient removal rate of the granules were also poor (de Kreuket al., 2005b). Aerobic granulation at low temperatures was notpossible with the present understanding.

2.2. Granule characterization

A large number of parameters were investigated to study char-acteristics of aerobic granules. These parameters include physical(settling velocity, density, specic gravity (SG), sludge volume index(SVI)), chemical (specic oxygen utilization rate (SOUR), EPS), and

those of smooth rigid spheres or of biolm-covered particles. Adavet al. (2008a) quantitatively evaluated the hydraulic characteristicsof aerobic granules using size exclusion chromatography. Theseauthors noted that the intra-granular transport for molecules largerthan approximately 140,000 Da can be severely limited by diffusionalresistance. On the other hand, Kim et al. (2008) highlighted thepossible aws using settling behavior to characterize the aerobicgranules at different COD loading rates.

2.3. Chemical parameters

Liu et al. (2003e) correlated the cell surface hydrophobicity andformationof heterotrophic andnitrifying granules. The hydrophobicityof granular sludge was two fold higher than the conventional bioocs(Liu et al., 2004a). Increase in shear force would increase the cellsurface hydrophobicity while cell surface hydrophobicity showedinsignicant effects on organic loading rates (Liu et al., 2003e). Detailedmechanisms corresponding to the noted changes remain unclear.

EPS are metabolic products accumulating on the surface ofbacterial cells, which could alter the physico-chemical characteristicsof cellular surface such as charge, hydrophobicity and other proper-ties. Adav and Lee (in press) extracted EPS from aerobic granules usingseven extractionmethods (Fig. 3). Since the aerobic granules exhibited

414 S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423biological parameters. General characteristics of sludge granules,such as size, structure, settling performance, stability against shearforces, EPS content, reactor performance, and metabolism rates werereported in research works (de Kreuk et al., 2005a; Etterer andWilderer 2001; Hulshoff Pol et al., 2004; Inizan et al., 2005; McSwainet al., 2004a; Moy et al., 2002; Tay et al., 2003).

2.2.1. Physical parametersSettling velocity of granules determined the efciency of solid

liquid separation which was a critical parameter for wastewatersymptoms. The settling velocity varied from 25 to 70 mh1 and wassignicantly higher than that of sludge oc (7 to 10 mh1) (Liu et al.,2003b; Qin et al., 2004b). High settling velocity increased the biomassretention capacity of the reactor and subsequently enhanced theorganic degradation capability. Mu and Yu (2006) evaluated the frac-tal dimensions of granules as an index to characterize the granulemorphology. Anuar et al. (2007) discussed the effects of mixingconditions on the settling behavior of aerobic granules. Mu et al.(2008) noted that the drag coefcients of aerobic granules were lessFig. 3. Flow chart of EPS extraction ma compact interior structure, different extraction methods yieldeddifferent extracted quantities of EPS. Adav and Lee (in press)recommended that ultrasound followed by the chemical reagentsformamide and NaOH outperformed other methods in extracting EPSfrom aerobic granules. The total polysaccharides/total proteins (PS/PN) ratios ranged between 3.4 and 6.2 for granules, much higher thanthat for sludge ocs (approximately 0.9). The relatively high proteinscontents presented an essential feature for cultivated aerobic granules(McSwain et al., 2005).

The excitationemission matrix (EEM) characterization of EPSdemonstrated three major peaks in the spectrum with excitation/emission (Ex/Em) wavelengths at 220230/340350 nm, [peak A]270280/340 350 nm, [peak B] and 330340/420430 nm, [peak C](Fig. 4). Based on the classication scheme developed by Chen et al.(2003), peaks A, B, and C were in regions II (aromatic proteins), IV(soluble microbial by-product-like), and V (humic acid-like), respec-tively. Sheng and Yu (2006) and Adav et al. (2007b) analyzed shifts inposition or change in intensity of EEM peaks as evidence of chemicalchanges in the extracted EPS.ethods (Adav and Lee, in press).

s. Gppliaxim

415S.S. Adav et al. / Biotechnology Advances 26 (2008) 4114232.3.1. Biological parametersScanning electron microscope (SEM), light microscopy, and CLSM

together with uorescence in situ hybridization (FISH) allowed in-sights into the microbial structure of the aerobic granules (Yi et al.,2003; Tay et al., 2002c; Jang et al., 2003; Meyer et al., 2003; Tsunedaet al., 2003). Heterotrophic, nitrifying, denitrifying, phosphorous-accumulating bacteria, and glycogen-accumulating bacteria have beenidentied in aerobic granules developed under different conditions(Jang et al., 2003; Meyer et al., 2003; Tsuneda et al., 2003; Lin et al.,2003; Yang et al., 2003). Microbial diversity of aerobic granules wasclosely related to the structure of aerobic granules and the composi-tion of culture media in which they were developed. The taxonomicmicrobial diversity in wastewater treatment plant and phenol-fed

Fig. 4. Excitationemission matrix (EEM) spectra for extracted EPS from aerobic granulewith mineral concentrations that were half those of Tay et al. (2001a,b). Aeration was suaeration, 30 min of settling and 4 min of efuent withdrawal. Incubation time=20 d. Maerobic granules showed the dominance of Proteobacteria (Jiang et al.,2004b; Whiteley and Bailey 2000; Snaidr et al., 1997).

Jiang et al. (2004b) identied ten isolates frommatured phenol-fedgranules of which six had taxonomic afliationswith -proteobacteria,three with Actinobacteria and one with -proteobacteria. Whiteleyand Bailey (2000) studied the bacterial populations in the speciccompartments of an operational industrial phenol remediationsystem using Proteobacterium group specic probes and found themajority to be - and -proteobacterium. Gram and Neisser stains andFISH analyses showed that most of the lamentous bacteria in aerobicgranules cultivated in brewery wastewater belonged to the genusThiothrix or to Sphaerotilus natans (Weber et al., 2007). FISH-CLSMtechnique identied that obligate aerobic ammonium-oxidizingbacterium Nitrosomonas spp. was mainly at a depth of 70 to 100 mfrom the granule surface, while anaerobic bacterium Bacteroides spp.was detected at a depth of 800 to 900 m from the granule surface(Tay et al., 2002c, Adav et al., 2007a,b,c,d,e). Toh et al. (2003) noteda layer of dead microbial cells at a depth of 800 to 1000 m. Adavand Lee (2008a) isolated nine strains and revealed the dominance of-Proteobacterium.

2.4. Granule applications

An examination of the applications of granules for wastewatertreatment showed many advantages: excellent settleability, high andstable rates of metabolism, resilience to shocks and toxins due to theprotection by a matrix of EPS, long biomass residence time, biomassimmobilization inside the aggregates and the possibility for bioaug-mentation. Bioaugmentation can be regarded as an effective tool inthe removal of xenobiotics from wastewater (Wuertz et al., 2004;Bathe et al., 2004).

2.4.1. Treating toxic organic wastewatersAerobic sludge granules have been successfully applied by Moy

et al. (2002) for treatment of high strength organic wastewater. Whenthe COD removal efciencies were stabilized, the potential of aerobicgranules to sustain high organic loading rates was examined by a step-wise increase in organic loading from 6 to 15 kg COD m3 d1 withoutcompromising granule integrity.

Phenol was considered to be toxic to aquatic species (Brown et al.,

ranules were cultivated with synthetic wastewater containing 1000 mg L1 phenol anded at a rate of 2 cm s1. The SBR was operated with 4 min of inuent lling, 202 min ofum granule size=1.2 mm.1967; Kibret et al., 2000; Chung et al., 2003) and added an odor todrinking and food-processing water (Rittmann and McCarty, 2001).Aerobic granules have been applied to degrade phenol (Jiang et al., 2002,2004a; Chou et al., 2004; Chou andHuang, 2005; Tayet al., 2005a,b). Tayet al. (2004b) demonstrated that the granules degraded phenol at aspecic rate exceeding1 gphenol g1 VSSd1 at 500mg L1 of phenol, orat a reduced rate of 0.53 g phenol g1 VSS d1 at 1900 mg L1 of phenol.Adav et al. (2007a) reported that aerobic granules could degrade phenolat 1.18 g phenol g1 VSS d1. One likely reason that accounted for thehigh degradation efciency of granules that degrade phenol was themass transfer barrier provided by a granule matrix that produced lowerlocal phenol concentrations on cells than the bulk value (Liu and Tay,2004).

Pyridine and its derivatives are by-products of coal gasication(Stuermer et al., 1982) and retorting oil shale (Leenheer et al., 1982),and were utilized as a catalyst in the pharmaceutical industry. Aerobicgranules could efciently degrade pyridine over initial concentrationsof 2002500 mg L1 (Adav et al., 2007a). At initial pyridineconcentrations of 250 and 500 mg L1, degradation kinetics followedclosely a zero-order kinetics with no time delay. The specic degra-dation rate of pyridine was 73.0 and 66.8 mg pyridine g VSS1 h1 at250 and 500 mg L1 of pyridine, respectively.

Phenol degrading granules have the capability of using bothphenol and pyridine. Adav et al. (2007a) applied MichaelisMentenkinetics and LineweaverBurk plot to study the behavior of granulesin the presence of phenol and pyridine and found competitive typeof interactions. The specic enzyme for pyridine degradation was

416 S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423present in the system that had a high afnity for phenol. Thus, phenolgranules could be applied for the removal of phenol in the presence ofpyridine in industrial wastewater.

The sludge granules were tested for removal of toxic organiccompounds by Xie (2003). Yi et al. (2006) reported on thebiodegradation of p-nitrophenol (PNP) by aerobic granules in a SBR.The specic PNP degradation rate increased with correspondingincrease in PNP concentration up to 40.1 mg L1 with a peak at 19.3mgPNP g of VSS1 h1, and declined with any further increase in PNPconcentration as substrate.

Wang et al. (2007b) efciently treated wastewater containing 2,4-dichlorophenol with glucose as a co-substrate. Zhang et al. (2008)noted that themethyl t-butyl ether (MTBE) can be efciently degradedby aerobic granular sludge with ethanol as a co-substrate.

2.4.2. Treating dairy wastewaterIt was well known that genuine wastewaters exhibited a rather

different behavior in biological degradability from that of syntheticwastewaters due to presence of a wide range and different type oforganic matters. Synthetic wastewater was used in almost all thestudies, but information on the performance of aerobic granules in thetreatment of genuine wastewater was rare (de Bruin et al., 2004;de Villiers and Ibala, 2003; Schwarzenbeck et al., 2004b). The dairywastewater contained signicant concentrations of nutrients andhence the removal efciency in this respect was also of great interest.Schwarzenbeck et al. (2005) used wastewater from dairy plant ininvestigating the performance of aerobic granules for dairy waste-water treatment. The removal efciencies of 90% of total COD, 80% oftotal N and 67% of total P were reported at a volumetric exchange ratioof 50%.

2.4.3. Removal of nitrogen and phosphorusComplete nitrogen removal involves nitrication and denitrica-

tion. Nitrite and nitrate produced from nitrication were reduced togaseous nitrogen by denitriers. Yang et al. (2004b) investigatedthe simultaneous removal of organics and nitrogen by aerobic gran-ules. This was due to the coexistence of heterotrophic, nitrifying, anddenitrifying populations in aerobic sludge granules. Similar hetero-trophic nitrifying microbial populations in aerobic granules andalternate nitrication and denitrication with nitrication rate up to97% and COD removal efciency of 95% were reported (Jang et al.,2003). Use of aerobic granules for nitrication and denitricationwere also reported by Beun et al. (2001). Dissolved oxygen con-centration had a pronounced effect on the efciency of denitricationby sludge granules.

Mosquera-Corral et al. (2005) revealed the signicant role ofoxygen on the simultaneous nitrication and denitrication pro-cesses. Low oxygen concentration resulted in a low rate of nitricationand a high rate of denitrication. Picioreanu et al. (1998) postulatedthat the low ratio of biomass growth rate versus diffusive transportresulted in the development of smooth biolms. If this proposed ratioalso determined the structure of the granules, selection for slowgrowing organismswould lead to stable granular sludge at low oxygenconcentrations. Performance of nitrifying granules that had beenproduced in aerobic upow uidized bed (AUFB) reactor in waste-water containinghigh concentration of ammonia (10001400 gNm3)had been studied (de Kreuk and Van Loosdrecht, 2004; Tsuneda et al.,2006). de Kreuk et al. (2005b) claimed that simultaneous COD, N, andP removal was possible at low oxygen saturation (20%), because ofheterotrophic growth inside the granules.

Phosphorus-accumulating microbial granules were cultivated atdifferent substrate P/COD ratios in the range from 1/100 to 10/100 inSBR by Lin et al. (2003). The granules had characteristic P-accumulatingpotentialwith concomitantuptake of soluble organic carbon and releaseof phosphate in the anaerobic stage, followed by rapid phosphate

uptake in the aerobic stage. The accumulated phosphorus showed adecreased trend with the increase in substrate P/COD ratio. A rangein granule P content between 1.9% and 9.3%, depending on the ratio ofP/COD of the inuent had been reported. Inuent with a P/COD ratio of2.5% resulted in granules with a P content of nearly 6%. Similar resultswere noted by Cassidy and Belia (2005) with an inuent P/COD ratio of2.8%. Removal of COD and P were over 98%, and removal of N and VSSwere over 97% in treating abattoir (slaughterhouse) wastewater withaerobic sludge granules in an SBR were reported by Cassidy and Belia(2005). Thayalakumaranet al. (2003) also reported similar COD,N, P, andVSS removals in an aerobic SBR treating a slaughter house wastewaterwith occulating sludge.

The simultaneous nitrication, denitrication and phosphorusremoval process was studied in a laboratory-scale SBR by alternateaerobic and anaerobic period for 450 days by Lemaire et al. (2007).Good phosphorus removal and nitrication occurred throughoutthe SBR with a dominance of Accumulibacter spp. (a polyphosphate-accumulating organism, PAO) and Competibacter spp. (a glycogennon-polyphosphate-accumulating organism, GAO). Accumulibacterspp. was dominant in the outermost 200 m region of the granulewhile Competibacter spp. dominated in the granule central zone.

2.4.4. Removal of heavy metals or dyesSeveral biomaterials includingmarine algae, fungi, activated sludge

and digested sludge were used as a biosorbent (Lodi et al., 1998;Taniguchi et al., 2000; Valdman and Leite, 2000). Major adsorbentscurrently used were in the form of suspended biomass (Liu et al.,2003c,d) but had several drawbacks such as post-separation, stabilityof biosorbents and regeneration after use. The maximum biosorptioncapacity of individual copper (II) and zinc (II) by aerobic granules wereclosely related to the initial concentrations of the copper and zinc inthe reactor and were 246.1 mg g1 and 180 mg g1 respectively (Xuet al., 2004). These drawbacks of suspended adsorbents were limitingtheir application for the removal of dyestuffs from industrial waste-water. Aerobic granules played a promising role in adsorption of toxicchemicals due to a high surface area, porosity and good settling ca-pability. The highly toxic heavy metals had been removed with sludgegranules by biosorption (Liu et al., 2002, 2003c,d). The removal of thecationic dye, Rhodamine B, by adsorption using aerobic granules wasgoverned by the Langmuir adsorption isotherm (Zheng et al., 2005).The maximum adsorption density of aerobic granules was three timesgreater than that of sludge ocs.

2.4.5. Removing particulate matterAerobic granular sludgewas successfully cultivated in a SBR treating

wastewater from the malting process with a high content of particulateorganic matter (Schwarzenbeck et al., 2004a). At an organic loadingrate of 3.2 kg COD m3 d1 and an inuent particle concentration of0.95 g L1 MLSS, an average removal of 50% in total COD and 80% insoluble COD could be achieved. A comparison of granular and occulentsludge grown under the same operating conditions showed no sig-nicant difference in removal efciency, although granules exhibiteda higher metabolic activity in terms of specic oxygen uptake rate.During the particulate removal by aerobic granulation, two distinctmechanisms were observed: during initial granule formation, particleswere incorporated into the biolm matrix and for mature granules, ahigh level of protozoa growth on the granule surface accounted for theability to remove particulate COD.

2.4.6. Removal of nuclear wasteThe use of aerobically grown granular biomass as novel biomaterials

for removing soluble uranium had been demonstrated by Nancharaiahet al. (2006). They studied the biosorption of uranium [U(VI)] at dif-ferent initial pH values (1 to 8) and different initial uranium con-centrations (6 to 750 mg L1) and observed rapid biosorption (b1 h) inthe acidic pH range (1 to 6) compared to that at pH 7.0 or above. Almost

1complete removal of uranium in the range 6100mg L in less than 1 h

was reported. The RedlichPeterson model gave the best t when theexperimental datawere analysed using different adsorption isothermequations. The maximum biosorption capacity of U(VI) was deter-mined to be 2182 mg g1 dry granular biomass. In Nancharaiah'sstudy, light metal ions such as Na+, K+, Ca2+ and Mg2+ were simul-taneously expelled from the granules, indicating involvement of anion-exchange-driven uranium uptake process. Uranium removalfrom very low strength solutions suggested that granular biomasscould nd application in the treatment of low-level liquid uraniumwastes, whereas other physicochemical methods were, either notsuitable or were expensive.

3. Granulation mechanism

3.1. Granulation steps

Studies have been proposed to examine the mechanisms that maycorrespond to the granulation processes. Microbial aggregation was ahypothesis in biology that occurred by intra-, inter- and multi-genericcell-to-cell attachment through cell surface receptors such as protein-saccharide or proteinprotein interaction (Kolenbrander 1985, 1999;Palmer et al., 2001). Tay et al. (2001a) recognized that aerobic

granulation was a process of microbial self-immobilization withoutcarrier support. Subsequently, Liu and Tay (2002) proposed that thefollowing four steps corresponded to the granulation process:

1. Microbe-to-microbe contact to form aggregates by hydrodynamic,diffusion, gravity and/or thermodynamic forces;

2. Initial attraction to form aggregates by physical (van der Waals, op-posite charge, thermodynamic forces), chemical (ionic pairing, tripletionic pairing, inter-particulate bridging) or biochemical (cell mem-brane fusion, cell receptor attraction, cell surface dehydration) forces.

3. Microbial forces to form aggregates by biological glue like cellularclustering and secretion of EPS.

4. Hydrodynamic shear force to stabilize the three dimensionalstructure of the granule.

5. Using CLSM coupled with different specic orochromes, uor-escent microspheres, and oligonucleotide probes, the interior ofbioaggregates collected during aerobic granulation process wasexamined (McSwain et al., 2005, Adav et al., 2007b,c). The CLSMimages for seed ocs and the mature granules using the multicoloruorescent technique recently developed by Chen et al. (2006,2007a,b) and Yang et al. (2007) are shown (Fig. 5). In brief, thedistributions of proteins, lipids, - and -polysaccharide, total cells

d inharidStep

417S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423Fig. 5. CLSM images of bioaggregates in different stage. The bioaggregates were cultivatestained for EPS [a proteins (green): FITC; b lipids (yellow): Nile red; c -polysaccSytox blue; f -polysaccharide (Blue): calcouor white]. (Step I) Un-occulated cells. (

granule core.sequential batch reactor with synthetic wastewater containing 500 mg L1 phenol ande (Light blue): Con A rhodamine; d total cells (red): SYTO 63; e dead cells (violet):II) Flocculated sludge oc of puffy structure. (Step III) young granule with dead cells at

418 S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423and dead cells were probed in the same bioaggregate. Theuorescent staining and CLSM tests demonstrated that microbialaggregation served as an initial step in granule formation (step IIin Fig. 5). The aggregated bacteria secreted EPS at the attachmentsite, multiplied and grew as large sludge ocs (step III, Fig. 5). Thesludge ocs then grew due to proliferation of the attached bacteriaand reunion forming granules. This granulation model took intoaccount the noted changes in morphology and interior structure.

3.2. Selective pressure driven hypothesis

In SBR operation, only particles that settle within a given timeframe could be retained in the reactor, while those with poor settle-ability were washed out from the system. Apparently, borrowed frombiological evolution theory, this physical screening step was con-sidered to provide a selection pressure to the biomass in the reactor,and only those which adapted to this challenge (to become big anddense enough to settle fast) would survive and be retained in thereactor (Liu and Tay, 2002). Tay et al. (2002b) studied nitrifyingbacterial granulation at different selection pressures and concludedthe need of strong selection forces for granulation. The requiredselection pressures had been created by keeping the constant columnheight and varying the discharge port height (Wang et al., 2004).Wang et al., (2007c) noted that the stability of the granule could beenhanced with stepwise increased selection pressure.

The physical settlingwashing out actionwas a pure screening stepwithout a demand for the microbes to respond to or to make changesupon the uid carryout, hence having different intended meaning bythe biological evolution theory. Microorganisms in high shearenvironments adhered by secreting EPS to resist damage of suspendedcells by environmental forces (Trinet et al., 1991). Experimental proofwas needed to justify how cells communicated with each other andtried to respond to the high liquid upow in order to become bigenough to remain in the reactor. Moreover, those tiny ocs that werewashed out initially had no chance to evolve with the environmentalchanges hence the selection is not fair. Nonetheless, the screening stepsuccessfully cultivated aerobic granules in SBR (Beun et al., 2002, Liuet al., 2005).

3.3. EPS enhanced granulation

Bacterium secreted sticky material called EPS constituting pro-teins, polysaccharides, humic acids, and lipids that assisted cell ad-hesion, thereby should be helpful to initiate the aerobic granulationprocess (Schmidt and Ahring, 1994; Tay et al., 2001c, Liu et al., 2004b).Accumulation of the secreted EPS correlated with the occurrence ofbiological adhesion and microbial aggregation (Costerton et al., 1981,Cammarota and Sant'Anna, 1998). Liu et al. (2004a,b) hypothesizedthat EPS bridged bacterial cells and other particulate matter into anaggregate. High polysaccharide content was noted to facilitate cell-to-cell adhesion and strengthen the microbial structure through a poly-meric matrix (Costerton et al., 1981; Tay et al., 2001b; Liu et al., 2002).Di Iaconi et al. (2006) noted that hydrodynamic shear compactedthe granules but did not affect the EPS content and compositions.On the contrary, metabolic blocking of exopolysaccharide synthesisprevented microbial aggregation (Cammarota and Sant Anna, 1998;Yang et al., 2004b). McSwain et al. (2005) reported high proteincontent in peptone and glucose-fed aerobic granules with a PN/PSratio of 6.610.9. Adav et al. (2007b) demonstrated that proteins,rather than polysaccharides, were enriched in the sheared granules,which was consistent with the results of McSwain et al. (2005) andChen et al. (2007a).

The EPS responsible for the structural integrity of aerobic granuleshas been argued to be non-readily biodegradable to its own producereven in the case of starvation (Sutherland, 1999), while other studies

showed that starvation was able to induce the EPS degradation bytheir own producers that caused bacterial detachment (Zhang andBishop 2003; Ruijssenaars et al., 2000). Bossier and Verstraete (1996)reported an induction of cell surface hydrophobicity due to starvation;yet a negative effect of starvation on cell surface hydrophobicity hadbeen reported by Castellanos et al. (2000). Conversely, constant cellsurface hydrophobicity was observed during carbon starvation (Saninet al., 2003; Sanin, 2003).

3.4. Granulation models

The hypothetical proposal by Liu and Tay's listsmost possible forcesthat corresponded to the granulation process. Mechanistic models onhow these forces really workwere still largely lacking. Amathematicalmodel developed by Ni et al. (2007) described the simultaneousautotrophic and heterotrophic growth in granule-based SBRs. Assum-ing that the autotrophs located at the rim regime and heterotrophs atthe core regime, this model predicted that the heterotrophs accountedfor most of the oxygen consumption than the autotrophs in thegranule. de Kreuk et al. (2007a,b) proposed a mathematical model foran aerobic granular sludge reactor that simultaneously removed COD,nitrogen and phosphate operated at SBRmode. Thismodel particularlydescribed the oxygen penetration depth into the granules, and theratio of anoxic and aerobic biomass, for overall nutrient removal.Xavier et al. (2007) proposed a multi-scale model of aerobic granularsludge SBR reactor by considering two-dimensional spatial arrange-ment of four bacterial groups: heterotrophs, ammonium oxidizers,nitrite oxidizers, and phosphate accumulating organisms (PAO). Thesimulations provided insight into the bioconversion processes withshort term dynamics and long reactor operation and integrated dy-namics of microbial metabolisms, diffusion reaction with 2-D spatialorganization. Ni and Yu (2008) and Ni et al. (2008) demonstrated thestorage and growth behavior of denitriers in aerobic granules usingactivated sludge models.

Up to now no proposed models reproduced the entire life for anaerobic granule, from its birth to death, with detailed description ofwhy a layered structure was formed and functioned in organic deg-radation. Restated, all models treated the substrate degradation, andcell growth and lysis in a simplied framework assuming no cellmigration. More works are needed to demonstrate this point.

4. A closer look

4.1. EPS and granule stability

One of the most serious barriers to practical applications of aerobicgranules was the loss of stability of aerobic granules over long-termoperation. Two different patterns were noted: granule break-up andlament overgrowth (Liu and Liu, 2006; Adav et al., 2007b, Zhu et al.,2008). For the former, the granules deteriorated into small pieces toow out with the upow liquid stream. For the latter, the outgrownlaments produced light and bulky granules for easy washout. Also,the laments tended to block the pipelines that led to failed reactors.Long-term operation was not possible without stable granules.

An example of the EPS and cell distributions in phenol-fed granuleis shown (Fig. 6). The -D-glucopyranose polysaccharides formed thecore, while the cells and -D-glucopyranose polysaccharides accumu-lated in the granule outer layers along with lipids. Wang et al. (2005b)applied only one dye, calcouor white, to their granules anddetermined that non-soluble -polysaccharide formed the outershell of aerobic granules to provide its strength. Conversely, McSwainet al. (2005) stained their granules using uorescein-isothiocyanate(FITC), concanavalin A (Con A) lectin conjugates and SYTO 63, to probethe content distribution of proteins,-polysaccharides and cells in thegranules. These authors and Zhang et al. (2007) argued that a non-cellular protein core in aerobic granule provided its stability. Based on

the results by Chen et al. (2007a), the Wang's and McSwain's groups

419S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423in fact highlighted only part of the whole story (Fig. 6). Stainingtechnique should be used with caution.

Adav et al. (2007d) selectively hydrolyzed proteins, -, -polysaccharides, and lipids using enzymes and determined thestability change following hydrolysis. These authors noted that,although protein was redundant at the core regime, the selectiveremoval of proteins had minimal impacts on the structural stability ofgranules. Conversely, hydrolysis of -polysaccharides caused granules'disintegration. The updated view was: the granule structure wasstabilized by a network principally composed of -polysaccharides asthe backbone for embedded proteins, lipids, -polysaccharides, andcells. Hence, enrichment of certain (not all) EPS assisted granulation,and enhanced granule stability. Such a conclusion was signicant toenhance granule stability during operation and to reduce granule lossin storage.

Storage reducedgranule stability (Tayet al., 2002c;ZhuandWilderer,2003). Tayet al. (2002c) andNg (2002) noted that thegranules stored foreight weeks became more irregular and smaller compared to freshgranules and released soluble organic material due to cell hydrolysis.The glucose-fed granules cultivated by Zhu andWilderer (2003) did notsignicantly change in size, color, or settleability after storage for sevenweeks at room temperatures.Meanwhile, Zhu (2004) claimed that their

Fig. 6. Phenol granule cultivated in sequential batch reactor with synthetic wastewatercontaining 250 mg L1 phenol, stained for all EPS components [proteins (green): FITC;B lipids (yellow): Nile red; C total cells (red): SYTO 63; D dead cells (violet): Sytoxblue; E -polysaccharide (light blue): Con A rhodamine; F -polysaccharide (Blue):calcouor white] and individual images were merged together.granules remained stable even after storage for two years in tapwater atan ambient temperature (1626 C). This result was too good to berealized in practice. Adav et al. (2007f) concluded that phenol-fedaerobic granules could be preserved better than acetate-fed granules atreduced temperatures. Particularly, when stored at sub-freezingtemperature (20 C), the granules could retain 8099% of the initialactivityafter 48h reactivation. Furthermore the additionof phenol in thestoring solution signicantly preserved the bioactivity of granules at allstorage temperatures. Chiu et al. (2007a,b) revealed that the cell corewas free of oxygen since the active cell layer, accumulated at the outerrim regime, consumed most intake oxygen. No oxygen was availableduring long-term storage. Adav et al. (2007f) probed obligate anaerobicBacteroides sp. over the entire interior of the granules stored at20 C for180 d. High storage temperature accompanied with the absence ofexternal substrate yielded endogenous respiration inside the granule.Restated, the EPS core could be digested inside out by the anaerobes.Following storage, theprotein core presented large vacuoles comparedwith the compact and solid protein core noted for fresh granules.

Low or even freezing temperatures and the presence of toxicsubstance (phenol) inhibited intra-granular bioactivity, hence assist-ing in preserving granule stability and cell viability for recovery.Furthermore the phenol-fed granule exhibited a dense -polysacchar-ide network in the granule interior, which explained why the latterpresented better stability than the former during storage.

4.2. Intra-granular transport

Chiu et al. (2006, 2007a,b) probed the DO proles around andinside a single aerobic granules. With the help of two DO microelec-trodes these authors established a DO proles under transient andsteady-state conditions. The external mass transfer coefcient aroundspherical oc or granulewas estimated by the Frossling equation. Chiuet al. (2006) estimated the apparent oxygen diffusivity (Dapp) ofacetate-fed and phenol-fed aerobic granules in the range between1.24 and 2.28109 m2 s1of 1.282.50 mm acetate-fed granules, and2.507.651010 m2 s1 of 0.420.78 mm phenol-fed granules byprobing the DO level at the granule center. It was noted that the EPScontent signicantly affected the intra-granular oxygen diffusivity.The combined multiple staining, CLSM and DO by microelectrode test,demonstrated an active layer of 125 m in thickness that consumedmost oxygen in the aerobic granules (Chiu et al., 2007a). The DO testscorrelated with the ndings by CLSM imaging results. The optimalgranular size for treating different wastewaters could be quitedifferent based on the intra-granular transport studies.

4.3. Strain interactions

Granules cultivated with inorganic carbon had a dominance ofammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB)(Holben et al., 1998; Hao et al., 2002; Jang et al., 2003; Tsuneda et al.,2003; Yang et al., 2004c). When granules were cultivated with acetateor glucose as a carbon source and nitrate as a nitrogen source, theidentied bacterial strains were the members of the genus Epistylis,Poterioochromonas, Geotrichum, Geotrichum klebahnii (Williams andde los Reyes, 2006). Yang et al. (2004a,b,c) cultivated aerobic granuleswith acetate as the sole carbon source and noted that successfulgranulation could be achieved only when free ammonia concentrationwas less than 23.5 mg L1. In addition, an increase in ammoniaconcentration signicantly decreased cell hydrophobicity and affectedthe EPS production, hence causing failure in granulation. Detailedresearch work on the mechanisms for ammonium inhibition and onthe possible inhibition by other metabolites or chemicals was still notfully understood.

Jiang et al. (2004b, 2006a,b, 2007) isolated ten bacterial strainsfrom aerobic phenol-degrading granules and identied their potentialfor degrading phenol. The PG-01 strain, a member of-proteobacteria,was common in granules and was the predominant strain in phenoldegradation. Another strain afliated with -proteobacteria, PG-08,had minimal phenol degradation capability, and a high propensityfor self-aggregation. Hence, different strains on aerobic granules mayhave specic roles in granule structural integrity and phenol degra-dation. Yeast genera, such as Rodotorula, Trichosporon, and Candidacould degrade high levels of phenol or phenolic compounds (Neujahr,1990; Kurtz and Crow, 1997; Chang et al., 1998; Ruiz-Ordaz et al.,2000; Chen et al., 2002; Alexievaa et al., 2004; Margesin et al., 2005).Jiang et al. (2005) isolated a Candida tropicalis strain from activat-ed sludge, and identied the growth kinetics and phenol-specicdegradation rates for this strain with phenol concentrations of 1002000 mg L1. Adav et al. (2007a) isolated the yeast strain, C. tropicalisfrom their phenol-degrading granules and reported it as a functionallydominant strain in the phenol degrading granules. Heinaru et al.(2000) isolated 39 bacterial strains from polluted river water (38Pseudomonas spp. and 1 Acinetobacter sp.) and found from the tax-onomic analysis of all the strains reported for phenol biodegradation,more than 70% strains belong to Proteobacterium phyla.

Adav and Lee (2008b) cultivated granules with a single strain,Acinetobacter calcoaceticus, which exhibited a high autoaggregation

potential with interconnecting brils (Fig. 7). Seed sludge with

s sha.

420 S.S. Adav et al. / Biotechnology Advances 26 (2008) 411423enriched Acinetobacter strains could form the granules faster than thesludge dominated by non-occulating microbial strain. The auto-

Fig. 7. TEM images showing the interactions of the strains (a) Acinetobacter calcoaceticuaggregation; (b) Bacillus thuringiensis with agellum; (d) Bacillus sphaericus with agellaggregating bacterial strains enhanced the granulation process(Adav and Lee, 2008b, Jiang et al., 2006a,b). The single-culturegranules cultivated with A. calcoaceticus degraded phenol at a rateof 993.6 mg g1VSS d1, signicantly higher than that of activatedsludge and of other aerobic granular sludge (Tay et al., 2005a;Watanabe et al., 1996). The different process performances noted withthe mixed-culture granules revealed the difculty in precise control ofthe dominant strains in the granules, partly because of the presence ofinhibiting strains. The single-culture granules could be biologicallyprecise for engineered systems. The strains A. calcoaceticus, Bacillusthuringiensis and Acinetobacter sp. were both phenol degraders andautoaggregators, and were capable of forming granules individuallywhen inoculated separately in a SBR (Adav et al., 2008b). Thesendings contradicted the previous view that autoaggregation andphenol degradation were mutually exclusive in aerobic granules andproposed trade off functional model (Jiang et al., 2004b, 2006a,b). Insingle culture granule, only one strain existed and the specic phenoldegradation rate could be higher than those frommulti-strains due toa lack of inhibiting strains, antibacterial substance and growthinhibiting metabolites. The interactions of C. tropicalis with A.calcoaceticus, B. thuringiensis suggested that the strains coaggregatedthrough the cell surface polymers by lectinsaccharide interactionswith the adhesin protein on A. calcoaceticus and complementary sugarreceptors on B. thuringiensis and C. tropicalis (Adav et al., 2008b). Thepresence of agellum on the B. thuringiensis and Bacillus sphaericushelped the nutrient current towards the granules as both these strainwere found to locate on the surface of the granules.

5. Summary and perspectives

Formation of granules in aerobic conditions has been possible andappears as a promising technique for high strength or highly toxicwastewater treatment. These granular systems allowed, in manycases, a more stable operation, and the treatment of larger loads,

owing interconnecting brils; (b) Bacillus thuringiensis and Acinetobacter calcoaceticusremoval of multiple toxic pollutants, inferior volumes for the settlingsystems and production of better quality efuents than any conven-tional systems. The formation mechanisms and applications andcertain recent efforts to explore this technology in depth has beenpresented. We also propose the following perspectives to the po-tential development of the aerobic granular sludge technology in thefuture.

5.1. Thermophilic aerobic granules

The literature regarding aerobic thermophilic applications wasreviewed by LaPara and Alleman (1999) and Guven (2004). Studieshave been conducted on thermophilic aerobic granular processeswhich have shown advantages over others including low wastebiomass production, higher degradation rates and reduced aerationbasin volume (Eckenfelder and Musterman, 1995), elimination ofcooling requirements for high temperature wastes (Tripathi, 1999),enhanced solubility and degradation of low-solubility substrate(Becker et al., 1999), and rapid inactivation of pathogens (LaPara andAlleman, 1999). Thermophilic aerobic processes could support auto-thermal operation or treatment of hot wastes from industries such aspulp and paper production (Barr et al., 1996; Tripathi and Allen, 1999;Jahren et al., 2002) and potato processing (Malladi and Ingham 1993).Operation of an SBR for cultivation of aerobic granules dominated bythermophilic microbial strain may be highly advantageous andcompetitive due to lower sludge production and pathogenic microbialstrains. At present, aerobic granulation system uses activated sludgethat contains many pathogenic microbial strains. Therefore, aerobicgranules without pathogenic microbial strain are needed for environ-mental and public health. This developing technology can assist inreducing contamination of receiving groundwater and drinking waterin pharmaceuticals and health care sectors.

421S.S. Adav et al. / Biotechnology Advances 26 (2008) 4114235.2. Combined processes

Granular bioreactors were able to couple with other treatmentunits to complement benets from both processes. For instance, Tayet al. (2008) proposed a new process, named aerobic granular sludgemembrane bioreactor (AGSBR) that combined membrane bioreactor(MBR) and aerobic granule technology for wastewater treatment. Thesystem indicated excellent membrane permeability with aerobicgranules in a four month operation compared with the conventionalMBR, and it was claimed that low membrane fouling was induced bythe high strength of the aerobic granules in the surface cake. Jun et al.(2007) also noted enhanced lterability for aerobic granular mem-brane over conventional biooc systems. These authors concludedthat the main components for fouling were proteins and polysacchar-ides in the water. The aerobic granular process has great potentialto work with anaerobic processes, pre-treatment/RO processes, andothers for utilizing its advantage of high biomass retention and tol-erance to toxicity from substrates.

5.3. Cultivation of aerobic granules with genetically engineered microbialspecies

The genes enabling degradation of environmental pollutants couldbe cloned into different bacteria. Many of the bacteria used to supplythe genes were actually discovered at hazardous waste sites, afternaturally evolving to cope with the hazardous chemicals present. Agenetically engineered bacterium that could reduce the concentrationof trichloroethylene by a thousand times has been reported byWinteret al. (1989). Genetically engineered bacteriumwith multiple requiredgenes could serve the purpose of removing multiple toxicants bysingle transformed bacterium. This area of work was new and hasgreater potential to be explored into further.

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