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Technical NoteReceived: 16 September 2010 Revised: 9 December 2010 Accepted: 5 January 2011 Published online in Wiley Online Library: 3 March 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2589

Start up of a pilot scale aerobic granular reactorfor organic matter and nitrogen removal1

Mariele Katherine Jungles,a,b Monica Figueroa,a Nicolas Morales,a

Angeles Val del Rıo,a Rejane Helena Ribeiro da Costa,b Jose Luis Campos,a

Anuska Mosquera-Corrala∗ and Ramon Mendeza

Abstract

Aerobic granulation is a promising technology for the removal of nutrients in wastewater. Since research to date is mainlyfocused at laboratory scale, a pilot-scale sequencing batch reactor (100 L) was operated to obtain granular sludge in aerobicconditions grown on acetate as organic carbon substrate. Selective pressure created by means of decreasing settling time andincreasing organic loading rate (OLR) enhanced the formation of aerobic granular sludge. Granules appeared after 6 days andreached an average diameter around 3.5 mm. The settling velocity value should be higher than 11 m h−1 in order to removeflocculent biomass. The reactor treated OLRs varying between 2.5 and 6.0 g COD L−1 d−1 reaching removal efficiencies around96%, which demonstrates the high activity and the ability of the system to withstand high OLR. Nevertheless, a rapid increasein the OLR produced a loss of biomass in the reactor due to breakage of the granules.c© 2011 Society of Chemical Industry

Keywords: aerobic granular sludge; granulation; organic loading rate; pilot reactor

INTRODUCTIONThe development of biomass in the form of aerobic granulesis under study for its application to the removal of organicmatter, nitrogen and phosphorus compounds from wastewateras an improvement to conventional activated sludge processes.1

Aerobic granular technology has several advantages comparedwith activated sludge processes, such as good biomass retention,ability to withstand shock and toxic loadings and presenceof aerobic and anoxic zones inside the granules to performsimultaneously different biological processes.2 The biomass growsas compact and dense microbial granules, enabling betterbiomass retention in the reactor, which is important for substrateconversion capabilities and for implantation. Aerobic granularreactors are able to treat high organic loading rates3 (OLRs), up to15 g COD L−1 d−1.

The key operational factors that promote aerobic granulationin sequencing batch reactors (SBR) have already been established,such as short settling times, large volumetric exchange ratioand short feeding periods.4 It was found possible to growgranular sludge in SBR reactors treating different kinds ofwastewaters5 and under different operational conditions ofoxygen limitation or low temperature.6 However, most of thiswork was carried out at laboratory scale, with only a few atpilot scale.1,7,8 Scale-up of granular systems leads to modificationof the hydrodynamic conditions, which are very importantfor the formation and maintenance of stability of aerobicgranules.

In the present work, the start-up and performance at differentOLRs of a pilot-scale aerobic granular SBR reactor were studied.During the last phase of the experiment, the stability of the systemwas tested by rapid changes in the applied OLR.

EXPERIMENTALExperimental work was performed in a bubble column reactor(total volume 125 L, working volume 100 L). The full reactor heightwas 177 cm, with working height 150 cm, diameter 30 cm, andliquid level after effluent discharge 75 cm. As a consequence thevolume exchange ratio (VER) was fixed at 50%. The effective heightto diameter ratio (H/D) was 5. Air was supplied (80–90 L min−1)through a ceramic dome provided with a fine bubble diffuser,placed at the bottom of the reactor. Peristaltic pumps wereused to feed the reactor through the top of the reactor (tapand concentrated synthetic feeding media) and to dischargethe effluent through a port placed at middle height of thereactor. A programmable logic controller (PLC) Siemens S7-224XP controlled the operational cycle, and also acquired online datafrom temperature, pH and dissolved oxygen (DO) sensors, whichwas stored in a PC using Siemens WinCC Flexible software.

The system was operated in cycles of 3 h distributed asfollows (min): feeding (7), aerobic reaction (165–168), settling(6–3) and effluent withdrawal (2). The hydraulic retention time

∗ Correspondence to: Anuska Mosquera-Corral, School of Engineering, Universityof Santiago de Compostela, Rua Lope Gomez de Marzoa s/n Spain.E-mail: anuska.mosquera@usc.es

1 Presented at Sustainable Solutions for Small Water and Wastewater TreatmentSystems Congress (S2Small2010), Girona 2010.

a School of Engineering, University of Santiago de Compostela, Rua Lope Gomezde Marzoa s/n Spain

b School of Environmental Engineering, University Federal de Santa Catarina,Campus Universitario. Caixa Postal: 476 - Trindade - Florianopolis- SC - CEP:88010-970- Brazil

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Table 1. Operational phases of the reactor over time

Phase DaysOLR

(g COD L−1 d−1)NLR

(g N L−1 d−1)COD : N

ratio

I 1–48 2.41 ± 0.26 0.14 ± 0.05 17.2

II 49–68 3.28 ± 0.35 0.29 ± 0.06 11.3

III 69–77 9.70–4.95 0.51–0.25 19.0–19.8

Table 2. Concentrated and trace solutions

Feeding media Trace solution

Compound Conc. (g L−1) Compound Conc. (g L−1)

CH3COONa· 3H2O 150.50 FeCl3· 6H2O 1.50

NH4Cl 18.49–22.70∗ H3BO3 0.15

K2HPO4 5.42 CoCl2· 6H2O 0.15

KH2PO4 4.28 MnCl2· 4H2O 0.12

MgSO4.7H2O 2.95 ZnSO4· 7H2O 0.12

KCl 2.47 NaMoO4· 2H2O 0.06

NaHCO3 12.34 CuSO4· 5H2O 0.03

CaCl2 1.22 KI 0.18

Trace solution 0.80 (mL L−1) EDTA 10

∗ In Phase II

(HRT) was fixed at 0.25 days. The reactor was operated atroom temperature (16–20 ◦C), without pH control, which rangedbetween 8.3 and 9.2, and with DO concentration always higherthan 5 mg O2 L−1. The system was inoculated with 3.7 g TSS L−1

of flocculent activated sludge collected from a municipal WWTPwhich included biological nitrogen removal characterized by asludge volumetric index (SVI) of 190 mL (g TSS)−1 and a VSS/TSSratio of 0.8.

The reactor was operated in three different phases during theoperational time depending on the applied organic and nitrogenloading rates (OLR, NLR) (Table 1). A concentrated syntheticfeeding solution was prepared according to the concentrationsshown in Table 2. Appropriate dilutions by tap water addition wereapplied corresponding to 1 : 100, 1 : 70 and from 1 : 50 to 1 : 25 inPhases I, II and III, respectively.

The pH, DO, nitrate, nitrite, ammonia, SVI and total and volatilesuspended solids (TSS, VSS) concentrations were determinedaccording to Standard Methods.9 The chemical oxygen demand(COD) concentration was determined according to Soto.10 Themorphology and size distribution of granules were regularlymeasured by means of an image analysis procedure11 using astereomicroscope ZEISS 2000-C.

Microbial populations were followed by the fluorescence in situhybridization (FISH) technique. Biomass samples from the reactorwere collected, disrupted and fixed with 4% paraformaldehydesolution.12 Bacterial cells were hybridized with the followingFISH probes: EUB338mix, ALF1b, BET42a GAM42a and NEU635.Details on oligonucleotide probes are available at probeBase.13

Fluorescence signals of disrupted samples were recorded withan acquisition system coupled to an Axioskop 2 epifluorescencemicroscope (Zeiss).

(a)

(b)

Figure 1. (a) Applied OLR (�) and COD removal efficiency (- - - - ) duringoperation of the pilot plant. (b) Concentrations of NH4

+-N in the influent(•), and NH4

+-N (◦), NO2−-N ( ) and NO3

−-N (�) in the effluent andnitrogen removal efficiency (- - - - ).

(a)

(b)

Figure 2. Profile concentrations of the different compounds during anoperational cycle: NH4

+-N (•), NO2−-N ( ), NO3

−-N (�), Total nitrogen(- - - - ), CODs (�) in (a) Phase I (day 35); (b) Phase III (day 77).

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Figure 3. Images of the biomass from the pilot-scale reactor at different operational days: (a) Day 6, (b) Day 9, (c) Day 27, (d) Day 37, (e) Day 63, (f) Day 77.The bar represents 2 mm.

RESULTS AND DISCUSSIONEfficiencies of organic matter and nitrogen removalThe pilot plant was started up applying an average OLR of 2.41 gCOD L−1 d−1 (Phase I, Fig. 1(a)). The removal organic matterefficiency quickly increased and, on day 6 of operation, it reacheda value around 92% and remained constant when an OLR of3.58 g COD L−1 d−1 was applied (Phase II). During Phase III, thepossible effects of OLR fluctuations on the reactor performancewere studied, first, by a sudden increase of the inlet OLR to 9.7 gCOD L−1 d−1 and then, by a decrease to 4.95 g COD L−1 d−1.Such changes had no effect on the organic matter removal andthe COD removal efficiency was around 96% during this phase. Itwas previously found14 that aerobic granular systems were able tomaintain an efficiency of organic matter removal higher than 96%when treating ORLs ranging from 6 to 12 g COD L−1 d−1.

During Phase I the average nitrogen removal efficiency wasaround 53% (Phase I, Fig. 1(b)). As neither nitrite nor nitrate wasdetected and considering the biomass growth during this period, itwas determined that the removed nitrogen was used for bacterialgrowth, as described elsewhere.15 This tendency was also observedduring Phase II until day 56, then nitrite and nitrate appeared inthe effluent. In Phase III, average nitrogen removal of around67% was achieved. It was determined that 17% of the nitrogen

was used for bacterial growth and the rest was eliminated vianitrification–denitrification. The nitrogen removal efficiency wasmaintained in spite of fluctuations in NLR, which would indicate thecapacity of the system to operate under different load conditions.16

Cycle profilesThe profiles of both COD and nitrogenous compound concentra-tions during a cycle were determined during phase I and III (Fig. 2).In Phase I (Fig. 2(a)), COD was consumed during the first 30 min ofthe cycle (23 min aeration, 14% of the reaction period) while am-monia slightly decreased due to biomass growth. During the restof the cycle ammonia concentration remained almost constant.Nitrification and denitrification processes started during Phase II(Fig. 1(b)). Denitrification was possible since part of the COD wasoxidized in the outer layers of the granules while another fractionwas used for denitrification in the inner layers during the feastperiod.17 Denitrified nitrogen oxides during this period came fromthe previous cycle. During Phase III (Fig. 2(b)) the consumptionrate for ammonia measured during the cycle was 101 mg NH4

+-N(g VSS d)−1. Once organic matter was depleted (after 25 min of aer-ation phase), nitrate and nitrite concentrations increased slightlyin the bulk liquid but total nitrogen concentration (TN: NH4

+-N +

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(a)

(b)

Figure 4. (a) Solids concentration in the reactor as TSS (•) and VSS (◦) andin the effluent as TSS (�) and VSS (�). (b) Evolution of the mean granulediameter with time.

NO2−-N + NO3

−-N) still decreased, which could indicate the oc-currence of denitrification using storage materials.18 Simultaneousnitrification–denitrification processes were observed in previousstudies where maximum N loadings of 0.26 and 0.24 kg NH4

+-Nm−3 d−1 were treated in a sequential batch airlift reactor19 andSBR,20 respectively.

Granule formation and propertiesThe reactor was started with a settling time of 6 min. With thislength of settling period and taking into account the depth ofthe discharge port (75 cm) the minimum settling velocity (Vsmin)for the biomass to be retained in the reactor is 7.5 m h−1. Smallgranules were observed after 6 days of operation (Fig. 3(a)), whilesuspended biomass used as inoculum was gradually washed out.Nevertheless, since the coexistence of these granules with a highamount of suspended biomass with worse settling properties wasobserved (Fig. 3(b)), the settling time was gradually decreased inorder to promote washout of that type of biomass. On day 20(Phase I), the settling time was changed from 6 to 4 min (Vsmin:11 m h−1) but flocs were still present inside the reactor (Fig. 3(c)).On day 36, it was shortened again from 4 to 3 min, resulting in aVsmin of 15 m h−1. Under such conditions, only granular biomasswas retained in the system (Fig. 3(d)). According to other authors,21

the value of Vsmin to enhance aerobic granulation should not beless than 8 m h−1. Due to the different reactor design in the presentwork the value of 11 m h−1 was not enough to obtain completeremoval of flocs from the system. As a result of the conditionsimposed on day 20 an initial decrease in biomass concentrationwas detected inside the system from 5.4 to 1.7 g VSS L−1, whichthen increased to 3.5 g VSS L−1 (day 42) (Fig. 4(a)). During PhasesI and II, biomass concentrations in the effluent were always lowerthan 0.2 g VSS L−1 and the estimated solids retention time (SRT)ranged from 2.8 to 22.0 d. The nitrification process started onlywhen SRT in the system was higher than 12 d. The SVI of thebiomass decreased from 190 to 26 mL (g TSS)−1 during the first

two phases. The mean diameter of granules gradually increasedand reached a value of 3.5 mm at the end of Phase II (Figs 3(e)and 4(b)). During Phase III, the applied OLR was increased to9.7 g COD L−1 d−1, which provoked breakage of the granules(Figs 3(f) and 4(b)), maybe due to mass transfer limitations, whichcaused a biomass concentration decrease to 3.4 g VSS L−1. Thisis in accordance with previous work3 where the compact regularmicrostructure of the acetate-fed granules was found to limit masstransfer of nutrients at an OLR of 9 kg COD m−3 d−1.

High solids concentrations up to 0.28 g VSS L−1 were detectedin the effluent (Fig. 4(a)) and the SRT decreased from 16.7 to2.8 d. The VSS/TSS ratio during the whole operating periodwas about 90–94%. It was previously observed that the appliedOLR had an important effect on the morphology and structuralproperties of the granules formed,22 therefore, high OLRspromote the formation of large and loose granules or eventhe disintegration of those formed.22 The food-to-microorganismratio (F/M) progressively decreased simultaneously with granulesformation. Due to the increase in VSS concentration the F/M ratiowas about 0.60 and 0.44 g COD (g SSV d)−1 in the final period ofStage I and during Stage II, respectively. In Stage III a sudden changein the OLR was applied, which provoked an increase of the F/M ratioto values near 2 g COD (g SSV d)−1. These F/M values were similar tothose in previous work (1.95 and 2.94 g COD (g MLVSS d)−1 whereno biomass granulation was observed.23 The F/M ratio appears tobe an important parameter affecting granules formation, as wasstated by Li et al.,22 who found that for sludge granulation the F/Mratio approached values of 0.5 g COD (g SS d)−1.

Microbial populationsThe FISH technique was applied to characterize the main bacterialpopulations present inside the pilot reactor in Phase II (Fig. 5).Probes ALF1b, BET42a and GAM42a were selected for theidentification of α-, β- and γ -Proteobacteria, respectively. Theapplication of probe EUB338I and DAPI in combination withprobe BET42a showed that an important part of the observedbacteria was β-Proteobacteria (Fig. 5(a)). The Alf1b and Gam42aprobes indicated the presence of a low percentage of α- andγ -Proteobacteria compared with DAPI (Figs 5(b) and 5(f)). ProbeNEU653, specific for the great majority of Nitrosomonas bacteriaspecies, gave positive results when nitrifying activity was observed(Fig. 5(c)). In this case, it can be observed that bacteria groupedin small clusters. Only a small proportion of the positive signalof β-Proteobacteria corresponded with Nitrosomonas. Duringobservation of the samples no type of filamentous bacteria wasdetected by application of the EUB338mix probe or DAPI.

CONCLUSIONSGranules have been formed in a pilot scale reactor, and stableoperating conditions of the system were reached, showing thatthis technology is suitable to obtain high efficiencies in termsof COD and nutrient removal. OLR and NLR varying from 2.5to 9.7 g COD L−1 d−1 and from 0.14 to 0.51 g N L−1 d−1 weretreated in the reactor with 96% and 67% removal percentages,respectively. The effect of rapid increase in the applied OLR from3.4 to 9.7 g COD L−1 d−1 provoked a breakage of the granules,which caused a decrease in biomass concentration. The selectionof an adequate settling time related with the Vsmin of the particlesis an important parameter in the SBR reactor. In order to obtaina high percentage of granular biomass the Vsmin imposed was

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Figure 5. FISH analysis images of a biomass sample from day 63 of operation. (a) β-Proteobacteria cells stained with Cy3–Bet42a (in red) and DAPI(in blue). (b) α-Proteobacteria cells stained with FITC–Alf1b (in green), γ -Proteobacteria cells stained with Cy3–Gam42a (in red) and DAPI (in blue).(c) Nitrosomonas-like cells stained with Cy3–Neu653 (in red) and DAPI (in blue). The bar represents 10 µm.

15 m h−1. This study provides a first approach to optimization ofthe operational conditions for further study of aspects such astreatment of industrial wastewater, how the formation of granulesand operating conditions are connected with changes in themicrobial structure, etc.

ACKNOWLEDGEMENTSThis work was funded by the Spanish Government(TOGRANSYS CTQ2008-06792-C02-01 and NOVEDAR Consoliderproject CSD2007-00055). Mariele Jungles thanks the BrazilianNational Research and Development Council (CNPq) for herscholarship (CT-HIDRO and SWE). The authors thank Mar Orge,Monica Dosil and Miriam Vieites for their support in the analyticaltechniques.

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