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Biochemical Engineering Journal 57 (2011) 7– 12

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journa l h omepage: www.elsev ier .com/ locate /be j

ultivation of aerobic granular sludge with a mixed wastewaterich in toxic organics

i Liua, Guo-Ping Shengb, Wen-Wei Lib, Zhong-Hua Tongb, Raymond J. Zengb, Jun-Xin Liuc,ie Xied, Shu-Chuan Pengd, Han-Qing Yub,∗

School of Earth and Space Sciences, University of Science & Technology of China, Hefei 230026, ChinaDepartment of Chemistry, University of Science & Technology of China, Hefei 230026, ChinaState Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, ChinaSchool of Resources & Environmental Engineering, Hefei University of Technology, Hefei 230092, China

r t i c l e i n f o

rticle history:eceived 27 April 2011eceived in revised form 18 June 2011ccepted 24 July 2011vailable online 30 July 2011

a b s t r a c t

Aerobic granular sludge was successfully cultivated in a sequencing batch reactor fed with a mixtureof chemical industrial wastewater rich in toxic organics and the effluent from an anaerobic acidogenicreactor. After 30-day operation, stable granules with a size of 1.0–3.0 mm were obtained. These gran-ules appeared to have rougher surface than those cultivated with the carbohydrate- or acetate-richwastewaters. There exhibited a “core” in the internal structure of the granules, which might benefit

eywords:erobic granulehemical industrial wastewaterequencing batch reactor (SBR)ludgeoxic substances

microorganisms to survive and resist the harsh environment. The formation of granules significantlyimproved the ability of sludge to withstand the toxic substances. The chemical oxygen demand removalefficiency of the granule-based reactor could reach around 80%, while its ammonia and total nitrogenremoval efficiencies reached 90% and 40%, respectively. The aerobic-granule-based reactor showed anability to resist the wastewater toxicity.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Aerobic granulation represents an innovative technology foriomass immobilization in biological wastewater treatment [1–4].n this process, the light and dispersed flocs are washed out gradu-lly, while the denser sludge particles are retained and accumulatedhrough a repetitive selection in sequencing batch reactor (SBR)perations, leading to the formation of compact and fast-settlingranules [5–8]. Such granule systems have been used to treat a wideariety of wastewaters, such as nutrient-rich diary wastewater9–11], soybean-processing wastewater [12], and textile wastew-ter [13]. Kishida et al. [10] cultivated granules using a syntheticastewater and through gradually adding the diluted livestockastewater into the influent, and used them for simultaneousitrogen and phosphorus removal from livestock wastewater.part from the frequently cited advantages, such as high biomass

etention, protection of microorganisms against predation andesistance to external disturbance, aerobic granules also show highesistance to toxic compounds. It has been demonstrated that gran-

∗ Corresponding author. Fax: +86 551 3601592.E-mail address: hqyu@ustc.edu.cn (H.-Q. Yu).

369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2011.07.005

ules were able to tolerate and degrade toxic organics such as phenol,p-nitrophenol and 2,4-dichlorophenol at levels that were knownto cause the breakdown of conventional activated sludge systems[14–16]. This is partially attributed to the unique structure of gran-ules, where the dense discrete microbial cells and extracellularpolymeric substances (EPS) matrix [17] set a barrier for mass trans-fer and lower the concentration of toxics on the inner cells.

The high toxic resistance and degradation ability of aerobicgranules are of particular interests for industrial wastewater treat-ment, which usually contains a large amount of toxic compounds.The cultivation of granules using toxic-containing wastewater hasbeen demonstrated in several previous studies [14–16]. However,synthetic wastewaters with a simple content of certain toxic com-pounds were generally used in these studies, while the cultivationof aerobic granules for treatment of complex toxic substance-containing wastewaters has not been reported so far.

To this end, a mixed wastewater composed of a chemical indus-trial wastewater rich in toxic organics and the effluent from ananaerobic acidogenic reactor was used for the cultivation of aerobic

granules in this study. The structural and biological properties of thecultivated granules were characterized, and the long-term perfor-mance of the granule-dominated reactor was investigated in termsof organic and nitrogen removals. This is an attempt to cultivate

8 L. Liu et al. / Biochemical Engineering Journal 57 (2011) 7– 12

Fig. 1. Images of sludge in the granulation process: (A) seeding sludge; (B) sludge on Daycross-section of a fresh granule (scale bar 1.0 mm).

Table 1Composition of raw chemical industrial wastewater and the mixed wastewater fedto the SBR.

Parameter Raw water (mg/L,except pH)

Influent to SBR(mg/L, except pH)

COD 3000–10,000 1000 (Stages 1 and 2)500 (Stage 3)NH4 500–5000 50TN 1000–10,000 100TSS 100–1000 <10TP <10 10pH 7.3 7.0

In Stages 1 and 2, the fraction of the raw chemical industrial wastewater in theifC

awt

2

2

etFtheiddos

TD

nfluent of the SBR was 10%. In stage 3, the fraction was increased to 1/3 and theraction of the UASB effluent was correspondingly reduced, resulting in an influentOD of 500 mg/L for the SBR.

erobic granules using a wastewater rich in toxic organics. Thisork presents a valuable effort in expanding the practical applica-

ion of aerobic granule technology in the wastewater treatment.

. Materials and methods

.1. Wastewater and reactor operation

The raw chemical industrial wastewater was the mixture of theffluents from a pesticide production plant, a chlor-alkali indus-rial plant and several other chemical plants which are located ineidong Chemical Industrial Park, Hefei, China. This chemical indus-rial wastewater was characterized by complex compositions, aigh microbial toxicity and a low biodegradability due to the pres-nce of various toxic organics. Moreover, significant fluctuationsn the water quality occur frequently, with the chemical oxygen

emand (COD) changing remarkably from 3000 to 10,000 mg/L inifferent months (Table 1). To facilitate microbial growth, the dosef a certain amount of nutrients into this wastewater is neces-ary. Thus, a mixture of this industrial wastewater and the effluent

able 2istribution of VFA (in unit of mM) in the anaerobic acidogenic reactor effluent.

VFA

Acetate Propionate i-Butyrate

3.97 ± 0.10 0.73 ± 0.07 0.18 ± 0.02

14; (C) granules on Day 30; (D) a single granule; (E) a freeze-dried granule; and (F)

from a laboratory-scale H2-producing upflow anaerobic sludge bed(UASB) reactor was used as the feedstock for sludge acclimation andgranule cultivation. This UASB effluent was chosen because it wasrich in volatile fatty acids, nitrogen and phosphorus [18]. The dis-tribution of VFA in the reactor effluent is listed in Table 2. The SBRoperation was divided into three stages: the sludge acclimation andgranule forming stage (about 30 days), the granule maturing stage(about 130 days) and the industrial wastewater treatment stage(about 140 days). At the first and second stages, the raw water wasdiluted, resulting in the influent COD of the SBR was approximately1000 mg/L, in which the chemical industrial wastewater had a frac-tion of 10%. At the third stage, the industrial wastewater fractionwas increased to a fraction of 1/3 and the fraction of the UASB efflu-ent was correspondingly reduced. And the influent COD was kept atapproximately 500 mg/L. The compositions of the raw wastewaterand the SBR influent are summarized in Table 1.

2.2. Seeding sludge and reactor set-up

The seeding sludge was taken from an aeration tank in Wangxi-aoying Municipal Wastewater Treatment Plant, Hefei, China. It hada mixed liquor suspended solids (MLSS) concentration of 2.6 g/L anda sludge volume index (SVI) of 74.2 mL/g. The specific gravity andthe settling velocity were 1.006 g/cm3 and 7.0 m/h, respectively.Experiments were carried out in a cylindrical column reactor withan internal diameter of 0.1 m and a height of 1.0 m (working vol-ume of 7.4 L and volumetric exchange ratio of 50%). Aeration wasprovided by means of air bubble diffusers at a volumetric flow rateof 500 L/h (1.77 cm/s of superficial air flow velocity), which gave aminimum dissolved oxygen concentration of above 2.0 mg/L. Thereactor was operated in an SBR mode with a total cycle duration

of 6 h, including: 3 min of feeding, 349 min of aeration, 1 min ofsettling (when granules were formed in the reactor), and 5 min ofeffluent withdrawal. The mean sludge residence time was 31 days.The reactor temperature was maintained at 25 ◦C using a belt heater

Butyrate Valerate Caporate

11.01 ± 0.10 1.74 ± 0.12 0.86 ± 0.04

L. Liu et al. / Biochemical Engineer

0 10 20 30 40 50 60

0

1

2 Soybe an-pr oce ssing wastew ater

Industr ial waste water Acetate

Mea

n di

amet

er (m

m)

Operati ng t ime ( days)

Fi

a7

2

t

F(

ig. 2. Changes of mean diameter of sludge in the granulation process of the chem-cal industrial wastewater-fed granules.

nd a temperature controller, and the reactor pH was in a range of.0 and 7.2.

.3. Analysis

The size of granules was measured using an image analysis sys-em (Image-pro Express 4.0, Media Cybernetics Inc., USA) with an

ig. 3. CLSM images of stained aerobic granule cultivated: (A) �-polysaccharides (Con Ascale bar 200 �m).

ing Journal 57 (2011) 7– 12 9

Olympus CX41 microscope and a digital camera (C5050, Olym-pus Co., Japan). The confocal laser scanning microscopy (CLSM)and environmental scanning electronic microscopy (ESEM) wereused to elucidate the structure characteristics of the cultivatedgranules. Granules for CLSM analysis were first fixed with 4%paraformadehyde in phosphate-buffered saline to minimize thestructural damages in the subsequent slice up process. Then, thegranules were stained using different fluorescene dyes of SYTO 63,calcofluor white and FITC, respectively. The staining details couldbe found in our previous paper [19,20]. The stained granules wereembedded for cryosectioning and frozen at −20 ◦C; after that, 20-�m sections were cut using a cryomicrotome and mounted ontothe microscopic slides for observation. CLSM (TCS SP2, Leica Gmbh,Germany) was used to visualize the internal structure of granules.In the acquisition of CLSM images, appropriate vision fields wereselected in order to collect the structural information for entiregranule. Granules were imaged with a 10× objective and analyzedusing Leica confocal software. Aerobic granules for the ESEM anal-ysis were prepared following the pretreatment method describedby Weber et al. [21]. The COD, ammonium, total nitrogen, MLSS,MLVSS and SVI were determined following the standard methods[22].

3. Results and discussion

3.1. Formation of the aerobic granules

The evolution of the sludge morphology in the granulation isillustrated in Fig. 1. The seeding sludge with a mean floc size of

); (B) total cells (SYTO 63); (C) proteins (FITC); and (D) combined image of (A)–(C)

10 L. Liu et al. / Biochemical Engineering Journal 57 (2011) 7– 12

0 50 100 150 200 250 300

0

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6

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I II III

Granule formed

Filamental thin fil ms were obser ved

(B)

ML

VSS

/ ML

SS

(C)

SVI (

mL

/g)

0psTguiscua

taawetso

Operating time (d)

Fig. 4. Biomass in the reactor: (A) MLSS, (B) MLVSS/MLSS, and (C) SVI.

.10 mm displayed a fluffy, irregular and loose-structured mor-hology (Fig. 1A). After 14 days of operation, fine bioparticlestarted to occur, indicating the initiation of granulation (Fig. 1B).hese bioparticles grew rapidly and, after 15 additional days, largeranules with diameters over 0.5 mm were observed (Fig. 1C). Grad-ally, granules with diameters of 1.0–3.0 mm became prevailing

n the reactor. The matured aerobic granules had a regular andpherical outer shape (Fig. 1D). Compared with the aerobic granulesultivated with synthetic wastewaters in other studies, the gran-les reported here exhibited a rougher surface and a bit yellowerppearance [12,23].

The changes of mean diameter in the granulation process forhe chemical industrial wastewater-fed granules with those for thecetate- [1] and soybean-processing wastewater-fed granules [12]re compared in Fig. 2. The formation of granules from seed sludgeas a gradual process, as evidenced by the increase in mean diam-

ter of the sludge. For the toxic industrial wastewater-fed reactor,he size of the primary particles increased faster than that for theoybean-processing wastewater-fed reactor, but was similar to thatf the acetate-fed reactor. After about 30-day cultivation, the mean

Fig. 5. (A) Images of thin-film surrounding the aerobic granular sludge and (B)translucent films stripped away from the granule (scale bar 1.0 mm).

diameter of the industrial toxic wastewater-fed granules reacheda plateau.

The image of a freeze-dried granule clearly shows that numer-ous holes of different sizes were distributed in the granules (Fig. 1E),which might facilitate the mass transfer within granules. It is inter-esting to note that a dark-color core was developed in the granuleinterior (Fig. 1F), and most of the granules in the reactor werefound to have such a core inside. In this process, microorganismswere firstly agglomerated into clusters and small bioparticles inthe harsh environments [24], and then gradually developed intomature granules after operation.

3.2. Morphology and structure of the aerobic granules

Fig. 3 reveals the CLSM images of �-polysaccharides (Con A),cells (SYTO 63) and proteins (FITC) in granules. The granulesshowed an intricate structure consisting of cell clusters (i.e., dis-crete aggregates of microbial cells in an EPS matrix) and manyinterstitial voids. According to the fluorescent intensity data pre-sented in the figures, the distribution of �-polysaccharides, cellsand protein were more plentiful in the core of the granule.

The use of CLSM in combination with different staining pro-tocols can provide a useful tool for visual investigation into thestructure of aerobic granules [17,19,20]. It is widely believed thatEPS play an important role in maintaining the structural and func-tional integrity of aerobic granules [17]. From Fig. 3, we can clearly

see that in the granule center there was a “core”, which was morecompact than the surrounding part of the granule. This was in goodagreement with the dark inner core observed in Fig. 1F. Such core-structure might be beneficial to the high toxicity-resistant ability of

L. Liu et al. / Biochemical Engineering Journal 57 (2011) 7– 12 11

F thin-g

tvTawagt

3

tibsgftsaTf0itptfii[wl

hwD

ig. 6. ESEM microstructure observation of thin-film adhered granule surfaces: (A)ranule surface; and (D) at high magnification.

he granules. Otherwise, cell clusters were separated by interstitialoids and channels, which created a characteristic porous structure.he aerobic granules had a compact core structure, which created

diffusion barrier and cells and bacteria in the granule interiorould encounter a lower concentration of toxic industrial wastew-

ter than those in the bulk liquid. In addition, the internal core of theranules stored proteins and polysaccharides, which strengthenedhe structure of the granules.

.3. Properties of the aerobic granules

Fig. 4 illustrates the changes of MLSS, MLVSS/MLSS and SVI inhe 300-day operation. With the acclimation of sludge, a rapidncrease in MLSS was witnessed at the first stage, indicating aiomass growth after adapting to the toxic wastewater. Since theettling time is a critical parameter and significantly influences theranulation process, a short settling time was generally consideredavorable for the granule formation [6,25]. In this study, a settlingime of 20 min was adopted in the initial three days to preventevere wash out of sludge. In Days 3–14, the settling period was sets 8 min, and was further decreased to 4 min after 14 days (Fig. 4A).he MLSS in the reactor increased from 2.5 to 4.12 g/L in the granuleorming process. The MLVSS/MLSS ratio was further increased up to.86 when granules were formed in the reactor (Fig. 4B). As shown

n Fig. 4C, the SVI value decreased to 29.8 mL/g rapidly, indicatinghat the sludge settling properties were gradually improved. Com-ared with the soybean-processing wastewater-fed granules [12],he SVI value decreased quickly in the granulation process of sludgeed with this toxic industrial wastewater and was much lower thant. Under the stress of the toxic industrial wastewater, microorgan-sms secreted more EPS, which accelerated the aggregation process26]. At the second stage, aerobic granules were adapted to the toxicastewater gradually, and both SS and SVI could be kept at a stable

evel.

Generally, inhibition of microbial growth tends to occur at a

igh toxic concentration. In the third stage, when the loading rateas increased, the MLSS was not affected immediately. But sinceay 221 a translucent thin-film started to form on the surfaces of

films linked to one another; (B) filamentous films; (C) cavities that appeared in the

some granules (Fig. 5A), which increased the viscosity of fluids inthe reactor. This film adhered to the granule surface and grew up insize gradually. When the granule size became larger, it peeled offfrom the granules and moved freely in the reactor (Fig. 5B). Suchan overgrowth of the filamentous thin-film led to the poor settlingability of the granules and their washout from the reactor [27]. Asa consequence, both SS and MLVSS/MLSS ratio declined to lowestlevels (Fig. 4A and B). Such a deterioration of sludge quality becameworse because of the shear force of water flow to granules. Fig. 6Aand B shows the ESEM images of the out surface of the film-adheredgranules. The filamentous films existed abundantly and connectedwith each other, forming cavities on the granule surface (Fig. 6Cand D). However, after Day 257, the settling ability of the gran-ular sludge was gradually recovered. Filamentous films and thoseattached granules were gradually washed out of the system and thenormal granules began to become the main stream of the reactor.And the SVI returned to the previous levels (Fig. 4C).

3.4. Reactor performance

The long-term performance of the reactor is illustrated in Fig. 7.At the first stage, the concentration of effluent COD decreased con-tinuously from 231 to 121 mg/L, indicating the adaption of thesludge to the toxic wastewater and the improved degradation abil-ity in this process. At the second stage, the granules were adaptedto the toxic wastewater gradually, and MLSS could be kept at arelatively stable level (Fig. 4A), the COD removal efficiency couldbe kept around 80% for a long time period (Fig. 7A). At the begin-ning of the third stage, when the industrial wastewater fractionin the influent to the SBR was increased, the COD removal effi-ciency decreased from 86% to 48%, but both MLSS and SVI of thesludge were not affected immediately (Fig. 4A). Gradually the CODremoval efficiency restored to its previous level. This indicates thatthe reactor had an ability to withstand the toxic substances. But

on Day 221, the COD removal efficiency began to decline becauseof the presence of filamentous-film granules in the reactor (Fig. 5).The COD removal efficiency was kept around 50% after the systemrecovered.

12 L. Liu et al. / Biochemical Engineer

0 50 100 150 200 250 3000

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TN

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ratio

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)

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removal (%

)

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[into aerobically grown microbial granules for the aerobic biodegradation of

ig. 7. Performance for the reactor with respect to: (A) COD; (B) ammonia; andC) TN. (�) Influent concentration, (�) effluent concentration, and (©) removal effi-iency.

The ammonia removal efficiency decreased to 14% on Day 14,nd then increased when granules were formed. The ammonia andhe total nitrogen removal efficiencies fluctuated obviously at therst and second stages, but at the third stage, the ammonia removalfficiency increased to around 90%. These results demonstrate theeasibility of cultivating aerobic granules with a toxic wastewaternd achieving effective treatment.

. Conclusions

Compact aerobic granules with a good settling ability were suc-essfully cultivated in an SBR using a wastewater rich in toxicrganics. After operating of 30 days, granules with a diameterf 1.0–3.0 mm were observed. The best reactor performance waschieved at Day 204 with a COD removal rate of 90.4%, an ammo-ia removal of 96.6%, and a total nitrogen removal of 59.9%. Theranules cultivated with such a toxic wastewaters exhibited a coretructure, which might benefit for resisting the harsh environment.s a result, the reactor exhibited the ability to withstand toxic sub-tances.

cknowledgements

The authors wish to thank the Natural Science Foundationf China (50738006 and 50828802), and the Key Special Pro-

[

ing Journal 57 (2011) 7– 12

gram on the S&T for the Pollution Control (2008ZX07316-003 and2008ZX07010-003) for the partial support of this study.

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