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International Biodeterioration & Biodegradation 95 (2014) 76e83

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Calcium effect on anaerobic biological treatment of fresh leachate withextreme high calcium concentration

Yan Dang, Rui Zhang, Sijun Wu, Zhao Liu, Bin Qiu, Yilong Fang, Dezhi Sun*

Beijing Key Lab for Source Control Technology of Water Pollution, Beijing Forestry University, P.O. Box 60, 35 Tsinghua East Road, Beijing 100083, China

a r t i c l e i n f o

Article history:Received 19 March 2014Received in revised form17 May 2014Accepted 20 May 2014Available online 16 June 2014

Keywords:Fresh leachateMSW incineration plantCalcium suppression thresholdCalcium precipitationEGSB reactor

* Corresponding author. Tel./fax: þ86 10 6233 6596E-mail address: sdzlab@126.com (D. Sun).

http://dx.doi.org/10.1016/j.ibiod.2014.05.0160964-8305/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Fresh leachate from municipal solid waste (MSW) incineration plant in China always contains extremelyhigh concentration of calciumwhich would reduce the bioactivity of microorganisms and lead to calciumprecipitation in granules. This paper investigated the effect of high concentration of calcium on anaerobicgranular sludge without calcium precipitation by static tests for 72 h, and on anaerobic bio-treatmentprocess for treating fresh leachate in a laboratory-scale expanded granular sludge bed (EGSB) reactorwith calcium precipitation for 132 d. The results showed that, suppression thresholds of calcium con-centration for anaerobic granular sludge were 5000 mg/L from both static tests and EGSB reactoroperation. Calcium precipitation in anaerobic granules was a gradual formation process, mainly in theform of calcium carbonate calcite. Compared with calcium precipitation, high calcium concentration wasmainly responsible for the decrease of COD removal efficiency in treatment process. Only a few micro-organisms survived when calcium concentration increased over 5000 mg/L, which was mainlyconcentrated in Clostridium of bacteria and Methanosaeta of archaea.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Municipal solid waste (MSW) incineration develops rapidly allover the world in recent years due to its advantages in MSWdisposal and energy production (Chou et al., 2009). In developingcountries, huge amount of fresh leachate from incineration plantsgenerates during the waste storage period (for 3e7 d) beforeincineration, because of the low calorific value and highmoisture ofMSW (Nie, 2008). This leachate needs to be treated individually aswastewater instead of spraying back to the incinerator furnaceswhich is commonly used in many European countries (Chen andChristensen, 2010). In the past decade, it has been frequentlyproved that anaerobic treatment was the most cost-effectivetechnology for the treatment of fresh landfill leachate (Agdag andSponza, 2005; Calli et al., 2006; Parawira et al., 2006;Kheradmand et al., 2010). However, the treatment of freshleachate from MSW incineration plants, the water quality of whichis different from fresh landfill leachate (Chen and Christensen,2010), are rarely reported.

Compared with landfill leachate, high calcium concentration(sometimes > 5000 mg/L) is one of the most typical characteristics

.

for leachate from MSW incineration plant, especially in developingcountries (Nie, 2008; Ye et al., 2011). This is mainly due to the largeamount of kitchen garbage without classification. Calcium inleachate may lead to calcium precipitation in granular sludge andscaling on the bioreactor wall and along effluent pipes, whichwould affect the operation of the reactor, and even cause accidentsin the actual projects (Van Langerak et al., 1998; Kim et al., 2003,2004). Furthermore, high concentration of calcium would also in-fluence the bio-treatment efficiency due to the calcium precipita-tion on granular sludge and the decrease of the biological activity ofthe microorganism (Yu et al., 2001; Liu et al., 2011). So far, there isno cost-effective way for high calciumwastewater pretreatment inreal project. And the high calcium wastewater, for example, freshleachate is usually directly transported into the bio-treatment unit.Therefore, it is important to investigate the calcium effect onwastewater treatment processes.

It has been reported that the calcium precipitation on sludgewould reduce the biomass activity (Yu et al., 2001). And thechannels in cell membrane for calcium ions transferring into mi-crobial cells are different from those for sodium and potassium ions(“calcium channel” at Dorland's Medical Dictionary, http://www.dorlands.com). Thus, the presence of high calcium concentrationin fresh leachate fromMSW incineration plant cannot simply equalto salinity caused by sodium and potassium. To the best of ourknowledge, studies on anaerobic bio-treatment of high calcium

Y. Dang et al. / International Biodeterioration & Biodegradation 95 (2014) 76e83 77

wastewater generally stayed at relatively low concentrations ofcalcium (about 1000 mg/L) for actual wastewaters (Van Langeraket al., 1998; Yu et al., 2001; Fern�andez-Nava et al., 2008), or highcalcium concentration for static tests or simulated wastewaters(Ahn et al., 2006). The suppression threshold of calcium concen-tration for bio-treatment of fresh leachate from MSW incinerationplant needs to be evaluated. Meanwhile, the effects of calciumconcentration and calcium precipitation on biomass activity ofanaerobic granular sludge are still unclear.

This paper investigated the suppression threshold of calciumconcentration and impact of calcium precipitation for anaerobicgranular sludge treating fresh leachate from MSW incinerationplant. Two set of experiments (static tests for 72 h and expandedgranular sludge bed (EGSB) reactor for 132 d) were carried out todemonstrate the effect of calcium concentration and calcium pre-cipitation. Calcium precipitation was characterized by scanningelectron microscopy (SEM), energy dispersive spectroscopy (EDS)and X-ray diffraction (XRD). The microbial communities in granularsludge samples throughout the operation process of EGSB reactorwere analyzed by denaturing gradient gel electrophoresis (DGGE).

2. Materials and methods

2.1. Fresh leachate

The fresh leachate used as the feed in this study was obtainedfrom an MSW-to-energy incineration plant in Beijing, China. Thecharacteristics of the leachate were shown in Table 1. Raw leachatewas diluted with tap water as influent, and then stored at 4 �C in arefrigerator.

2.2. Laboratory-scale EGSB reactor treating fresh leachate

The suppression threshold of calcium concentration togetherwith impact of calcium precipitation for anaerobic granular sludgetreating fresh leachate from MSW incineration plant was investi-gated in a laboratory-scale EGSB reactor. The reactor, the schematicof which was shown in the previous study by Dang et al. (2013)with working volume of 4.5 L, was operated stably at 33 ± 1 �Cthroughout the study. Internal recirculation was applied and theliquid up-flow velocity was maintained at 2.0 m/h. The anaerobicgranular sludge inoculated into the EGSB reactor was 6.6 g VSS/L(volatile suspended solids), taken from a full-scale UASB reactorusing to treat brewery wastewater in Henan, China, with VSS/TSS(total suspended solids) of 0.72. The hydraulic retention time (HRT)was kept at 2.5 d, and the sludge retention time (SRT) was between32 d and 49 d.

The fresh leachate was dilutedwith tap water as feed of the EGSBreactor. In order to avoid the influence of organic load and highconcentration free ammonia, we diluted the leachate till the COD ofwhich was about 17,000 mg/L so as to ensure the COD and ammonia

Table 1Characteristics of the leachate from the MSW incineration plant (unit: mg/L, exceptfor pH).

Item Value Item Value

COD 70,390e75,480 Ca 3275e5827BOD5 39,250e46,458 Na 2273e3728NH4

þeN 1042e1395 Mg 463e1598TN 1330e2179 Fe 59.1e679.9TP 104.6e163.8 Mn 4.56e43.50Cl- 3978e4729 Zn 13.9e36.5SO4

2� 1833e2907 Pb 1.11e7.61pH 4.58e6.42 Ni 0.91e2.3

concentrations (free ammonia concentration was in the range of23e32 mg/L in the reactor) were lower than the suppressionthresholdmentioned in other studies (Ye et al., 2011; Liu et al., 2012).After anaerobic sludge acclimated to the diluted leachate composi-tion (operating for 1.5e2 SRT), keep the leachate concentration ininfluent and gradually increase the calcium concentration by addingcalcium chloride to investigate the calcium effect on the treatmentprocess in EGSB reactor. Wang et al. (1995) reported that no inhi-bition effect on the anaerobic system occurred when the accumu-lated Cl� concentration was less than 20,000 mg/L.

2.3. Static tests

The suppression threshold of calcium concentration for anaer-obic granular sludge in static tests was measured by specificmethanogenic activity (SMA). The sludge was withdrawn from theEGSB reactor treating fresh leachate at the end of start-up period(without calcium precipitation). Take the same weight of sludgeinto a series of 125-mL serum bottles with media (acetic acid15,000 mg COD/L, NH4Cl 1700 mg/L, KH2PO4 370 mg/L, MgSO4

90 mg/L, trace elements 0.1 mL, Na2S solution 1000 mg/L yeastextract 2000 mg/L, pH 7, Ahn et al., 2006) and different concen-trations of calcium (0 mg/L, 500 mg/L, 1000 mg/L, 2000 mg/L,3000 mg/L, 5000 mg/L, 8000 mg/L) under strict anaerobic condi-tions and incubate them inwater bath shaker at 35 �C and 120 rpm,following the procedure reported by Fang et al. (1995). The SMAanalyses were conducted in triplicate for each calcium concentra-tion. Methane productionwas measured every certain hour using aglass gas displacement device filled with 3 mol/L of NaOH solution.SMA was calculated from the linear range of the specific methaneproduction rate curve using linear regression. The degrees of in-hibition of SMAs under different calcium concentration are usingspecific methanogenic activity ratio (SMAr) to determine, as shownin equation (1).

SMAr ¼ SMAs=SMAk � 100% (1)

where SMAs stands for the SMA of granular sludge under differentcalcium concentrations, SMAk stands for the SMA of granularsludge under no calcium addition media, which is called blank.

2.4. Analytical methods

COD, BOD5, TSS, VSS, total N (TN), total P (TP), and NH4þ-N were

determined using standard methods (APHA et al., 1998). Volatilefatty acids (VFAs) were measured by titration (Anderson and Yang,1992). pH was measured with a Thermo Orion 3-Star pH meter.Calcium concentrations were analyzed using a Dionex-4500i IonChromatogram with an IonPac AS14 column.

2.5. SEM and EDS analysis

For SEM and EDS analysis, granules were fixed for 2 h in 2.5%glutaraldehyde. After rinsing twice with sodium cacodylate buffer,the granules were fixed for 1.5 h in 1% osmium tetroxide. Afterrinsing with demineralized water, the aggregates were dehydratedin an ethanol series (10, 30, 50, 70, 90 and 100%, 20 min per step)and subsequently critical-point dried with carbon dioxide. At last,the aggregates were coated gold/palladium sputter, before exam-ining on a SEM (JSM6300F, Jeol).

2.6. XRD analysis

XRD patterns were recorded over a 2 h from 10� to 90� in a scanrate of 3�/min using nickel-filter Cu Ka radiation (l ¼ 0.15418 nm)

Fig. 1. Performance of EGSB reactor at start-up period.

Y. Dang et al. / International Biodeterioration & Biodegradation 95 (2014) 76e8378

with a graphite monochromator at 40 kV and 120 mA (D/MAX-RB,Rigaku, Japan). The sludge samples were washed thoroughly by de-ionized water, dried at 105 �C and ground before analysis.

2.7. DNA extraction and amplification

Sludge samples werewithdrawn for about 1 g each (wet weight)from the EGSB reactor and washed several times with the phos-phate buffer solution and then mechanically bead beaten with amini-beadbeater (Biospec, USA). Then, total DNAwas extracted andwas purified using a FastDNA kit for soil following the manufac-turer's instructions (Q-BIOgene, CA, USA). Different primer pairs(Ovreas et al., 1997; Grobkopf et al., 1998) were applied during thePCR amplification. The V2eV3 region in 16S rRNA fragments ofarchaea were amplified using the primer pair PRUN109F (50-ACKGCTCAGTAACACGT-30) and PRUN519R (50-TTACCGCGGCKGCTG-30), with a GC clamp attached to the 50 end of the reverse primerto enable the separation of the fragments, whereas the primer pairEUB338F (50-ACTCCTACGGGAGGCAGCAG-30) and EUB534R (50-ATTACCGCGGCTGCTGG-30), with a GC clamp attached to the 50 endof the forward primer, was applied to amplify the V3 region in 16SrRNA fragments of eubacteria. The PCR conditions were as follows:predenaturation at 94 �C for 5 min; 25 cycles of denaturation at94 �C for 45 s, annealing at 55 �C for 30 s, and elongation at 72 �C for1 min; and then post-elongation at 72 �C for 10 min. The ampliconswere stored at 4 �C for further analysis.

2.8. DGGE analysis and sequencing

The PCR products were separated via DGGE using the BioRadDCode™ Universal Mutation Detection System (BioRad, USA). ThePCR products were applied to 8% (m/v) polyacrylamide (37.5:1acrylamideebisacrylamide) gels in 1� TAE buffer containing35e55% denaturing gradient for archaea and 35e65% denaturinggradient for eubacteria. The 100% denaturing solution contained7 M urea and 40% (v/v) formamide. Electrophoresis was performedin 1� TAE buffer at 70 V and 60 �C for 15 h. The DNA was stainedwith SYBR Green I dye (Sigma) and visualized under UV light.

The similarity and diversity of the microbial community wereevaluated using Quantity One (BIO-RAD 4.6.2) and the Shannondiversity index (SDI) (Shannon and Weaver, 1963), respectively.

The dominant DGGE bands of every profile were excised fromthe gels with sterile scalpel, and washed with TE buffer in sterileEppendorf tubes. The DNA was re-amplified using the Primerspairs, PRUN109F and PRUN518R (with no GC clamp) for archaea,EUB338F (with no GC clamp) and RUB534R for Eubacteria. Forcloning, 16S rRNA gene amplicons were purified with a Cycle-PureKit (Omega, USA). And the purified amplicons were ligated to thevector then were cloned and ampicillin selection and blue/whitescreening were applied to select for positive clones. The selectedclones were sequenced and compared to NCBI database using theBLAST search option. The phylogenetic trees were also constructedby the neighbor-joining method using MEGA 5. The stability of thephylogenetic trees was analyzed by Bootstrap (1000 replicates).

The sequences obtained in this study are available in GenBankunder accession numbers KJ574119-KJ574137 (release date on April11, 2014).

3. Results

3.1. EGSB reactor start-up and anaerobic granular sludgeacclimation

The EGSB reactor start-up period was lasted for 70 d. Theleachate concentration in influent gradually increased till COD of

~17,000 mg/L and NH4þeN of ~300 mg/L. And then, the reactor was

operated at this leachate concentration stably for 40 d to let thegranular sludge acclimated to the leachate composition. The CODremoval efficiency was remained between 95.0% and 98.2% at thisperiod except for the first 5 d (Fig. 1a), and the VFAs was less than8 mmol/L consistently (Fig. 1b), while the alkalinity in reactorgradually increased to about 11,000 mg/L (CaCO3 calculated). Theseindicated that the start-up of EGSB reactor was successful andefficient (Ahring et al., 1995). The calcium concentration in influentwas about 500e1000mg/L. When it came to the end of the start-upperiod, the VSS/TSS was 7.0, which indicated that very little calciumprecipitation occurred in the sludge within 70 d of operation undercalcium concentration less than 1000 mg/L. The sludge samplesusing in the static tests were withdrawn from the reactor at the endof the start-up period.

3.2. Suppression threshold of calcium concentration for anaerobicsludge in static tests

The SMAs of acclimated anaerobic granular sludge samples wereinvestigated in static tests with different calcium concentrations for72 h. From the experimental results (Fig. 2), it clearly showed thatwhen calcium concentration was 500 mg/L, the methane produc-tion was much higher than that of blank, the SMAr was 314.85%. Itindicated that low calcium concentration can promote the meth-anogenic activity of granular sludge, which was similar to theresearch result reported by Ahn et al. (2006). However, when cal-cium concentration increased to 1000mg/L, the calcium promotioneffect was not obvious, and the SMAr was reduced to 123.43%. And

Fig. 2. SMAr of anaerobic granular sludge under different calcium concentrations instatic tests.

Y. Dang et al. / International Biodeterioration & Biodegradation 95 (2014) 76e83 79

when calcium concentration increased from 2000 mg/L to3000 mg/L, the methane productionwas a little bit less than that ofthe blank, the SMAr was between 89% and 95%. It indicated thatunder these calcium concentrations, the SMAs of granular sludgewas slightly inhibited. Further increase calcium concentration to5000 mg/L and 8000 mg/L, the SMAr significantly decreased to32.45% and 14.54%, respectively, which was belonging to severeinhibition (Ahn et al., 2006). The VSS/TSS values of all sludgesamples were analyzed, and the results showed that the valueswere all about 0.690 ± 0.005, which were almost the same to thevalue of VSS/TSS sludge at sampling time, indicating that therewere almost no calcium precipitations on granular sludge in 72 h ofstatic tests. It can be concluded that the calcium suppressionthreshold for anaerobic sludge was 5000 mg/L in static tests.

3.3. Suppression threshold of calcium concentration for anaerobicsludge in EGSB reactor

The long time operation for calcium suppression thresholdtogether with calcium precipitation was investigated in thelaboratory-scaled EGSB bioreactor. When the start-up periodfinished, the influent leachate concentration was remained at COD~17,000 mg/L, and the calcium concentration in influent wasgradually increased from 500mg/L to 7000mg/L by adding calcium

chloride from 71st d to 132th d. The performance of EGSB reactor atthis period was shown in Fig. 3. When calcium concentrationincreased from 500 mg/L to 4000 mg/L, the EGSB reactor was stilloperated in a good and efficient status although the COD removalefficiency slightly decreased from over 98% to about 96%. Whencalcium increased to 5000 mg/L, the COD removal efficiencydropped to 93% at first, and gradually decreased to 89% after 16 d ofoperation. It meant the inhibition of calcium effect was observedobviously. Further increase calcium concentration to 6000 mg/Land 7000 mg/L, the COD removal efficiency declined to <60%within 13 d. It indicated that the bioactivity was severe inhibitedunder these calcium concentrations, and the leachate treatment ofEGSB reactor was collapsed.

The VFAs, pH and alkalinity in effluent were also monitoredduring rising calcium concentration period. The VFAs in effluentwas constantly less than 10 mmol/L when calcium concentrationwas <4000 mg/L before 97th d. However, when calcium concen-tration increased from 5000 mg/L to 7000 mg/L, the VFAs ineffluent dramatically increased from 9.6 mmol/L to 56.0 mmol/L(Fig. 3b). The VFAs increasing also resulted in the pH decline ineffluent (Fig. 3c). Meanwhile, alkalinity in reactor decreased from11,700 mg/L to about 2000 mg/L (CaCO3 calculated).

3.4. Calcium precipitation in anaerobic granules

From the calcium concentration in influent and effluent of EGSBreactor at different period (Table 2), it can be easily found thatmuch calcium precipitated in the bioreactor in some form. And theVSS/TSS value gradually decreased from 0.690 to 0.129 with theincrease of calcium concentration, indicating the biomass in sludgeslowly decreased, which demonstrated that the calcium precipi-tation was a slow formation process.

SEM images of the granules show that (Fig. 4), the seed sludgewas much smaller than the granules withdrawn from the EGSBreactor on 132nd d. From the sectional view, the internal of seedsludge was full and uniformwith rich microbial of cocci and bacilli.However, it was interesting to find that, the internal of the granularsludge from 132nd d appeared large cavity, which due to the cal-cium precipitation.

The EDS analysis showed that (picture and table not shown, findthe Supplementary materials), the calcium precipitation wasmainly concentrated in the internal of granular sludge. In the centerof the granular sludge from 132nd d, calcium content was up toover 30% among the major elements.

The XRD analysis revealed that calcium precipitation in granularsludge was mainly in the form of calcium carbonate calcite (figurenot shown, find the Supplementary materials), which was the sameas the research result reported by Yu et al. (2001).

3.5. Microbial community

Sludge samples were collected from the middle part of the re-action zone in EGSB reactor on 0 d, 70 d, 78 d, 104 d, and 132 d ofoperation. The sludge sample from 0 d was the seed sludge, and thesample from 70th d was the acclimated sludge. The samples from78 d, 104 d, and 132 d were withdrawn from the reactor whencalcium concentration in influent was 2000 mg/L, 5000 mg/L and7000 mg/L, respectively.

From the DGGE fingerprint (Fig. 5) together with similarity anddiversity analysis (tables not shown) for both archaea and eubac-teria, it can be concluded that the similarity and diversity for botharchaea and eubacteria were all gradually decreased with the cal-cium concentration in influent increasing. Many microorganismsrepresented by certain bands (e.g. bands C, D, E, F, G for archaea andbands 6, 8, 10, 11, 12 for eubacteria) were gradually disappeared

Fig. 3. Performance of EGSB reactor in rising calcium concentration period.

Table 2Calcium concentration in effluent and VSS/TSS at different calcium influent.

InfluentCa2þ (mg/L)

500 2000 3000 4000 5000 6000 7000

Effluent Ca2þ

(mg/L)26.827 45.233 143.068 495.149 806.953 2118.622 3398.988

VSS/TSS 0.690 0.627 0.573 0.496 0.347 0.274 0.129

Y. Dang et al. / International Biodeterioration & Biodegradation 95 (2014) 76e8380

with the operation of reactor, indicating that the survivals of thesemicroorganisms were limited by the leachate composition or thehigh calcium concentration. The phylogenetic affiliations of mainDGGE band sequences were summarized in Table 3 (phylogenetictrees were not shown, find the Supplementary materials).

4. Discussion

With calcium concentration of 5000 mg/L, the reduction oftreatment efficiency for EGSB was not as much as that of SMA forgranules in static tests. This is because the impact resistance oflarge scale reactor was much stronger than that of small scaleexperiment (125 mL serum bottle). In addition, the large amount ofsludge in EGSB reactor would avoid COD removal from decliningdramatically. However, although COD removal efficiency wasremained high, the inhibition of calcium effect was observedobviously, as the treatment efficiency decreased continuouslywithout stably. From this point of view, the calcium suppressionthreshold for bio-treatment process in treating fresh leachate wasregarded as 5000 mg/L.

When calcium concentration increased over 5000 mg/L, VFAs ineffluents increased dramatically. It could be inferred that therewere many small molecule acids accumulated in the bioreactorwithout transferring to methane under high calcium concentrationcondition, which might be caused by the calcium inhibition formethanogens' activity. At the same time, alkalinity in effluent wasalso decreased significantly, indicating the impact resistance of thereactor was weakened at high calcium concentration. The reduc-tion of alkalinity was mainly because the produced CO2 from theanaerobic degradation and CO3

2� and HCO3� in the reactor reacted

with the arising Ca2þ in influent and formed calcium carbonate (Yu

et al., 2001; Ye et al., 2011). The sharp increase of VFAs and decreaseof alkalinity in effluent leading to a high ratio of VFAs/alkalinity (upto 1.24). Anaerobic processes can be considered to be operatingfavorably without acidification risk when the VFAs/alkalinity ratiois less than 0.3e0.4 (Leit~ao et al., 2006). The high VFAs/alkalinityratio suggests that the buffering capacity in the reactor was poorand did not suffice to maintain a proper pH level, which could havefurther inhibited the methanogenesis.

After 132 d of operation in EGSB reactor, calcium precipitationoccurred in granular sludge, which affected the methanogens' ac-tivity. On one hand, the calcium precipitation in granules affectedthe mass transfer of methane and other bio gases, further formedthe cavities in granules, affecting the methane activity of granules(Liu et al., 2011). On the other hand, the calcium precipitationaffected the mass transfer process fromwastewater into the sludge(Kettunen and Rintala, 1998), reducing the contact of organics withmicroorganisms, further affecting the treatment effect.

Calcium precipitation in anaerobic granules was a gradual for-mation process. The appearance of calcium precipitation impact onSMA of granules also needed a long operating time under highcalcium concentration condition. The calcium suppression thresh-olds for anaerobic sludge, the experimental results of which

Fig. 4. SEM photographs of anaerobic granules from EGSB reactor (a, b, c represented seed sludge, d, e, f represented the granules from 132nd d).

Y. Dang et al. / International Biodeterioration & Biodegradation 95 (2014) 76e83 81

obtained from both static tests without calcium precipitation andEGSB reactor process with calcium precipitation, were almost thesame. Thus, it can be drawn a conclusion that, high calcium con-centration was the main cause of COD removal decrease for treat-ment process, compared with calcium precipitation.

Microbial community analysis showed that, for eubacteria,bacteria represented by bands 6, 8, 10, 11, 12 gradually disappearedwith the increasing of calcium concentration, indicating that thesurvivals of these microorganisms were limited. The bacteria

Fig. 5. DGGE fingerprints of anaerobic sludge from different operation period

represented by band 6 and band 8 were related to Anaerovorax sp.(JN692194) and Chloroflexi bacterium (DQ330295), respectively.Anaerovorax can utilize putrescine to acetate, butyrate, molecularhydrogen and ammonia (Matthies et al., 2000). These bacteriawereall strictly anaerobic, gram-positive, non-spore-forming bacteria.The absence of these bacteria under high calcium concentrationmay lead to COD removal dropped.

Bacteria represented by bands 1, 2, 3, and 4 were the dominantbacteria in the sludge during the whole research period. The

s (frame a represented the archaea and frame b represented eubacteria).

Table 316s rRNA gene sequence-based phylogenetic affiliations of main DGGE bands using aBLAST search of the NCBI database.

Band Phylum, class,or order

Closest species or taxon(accession number)

Similarity(%)

1 Chloroflexi Uncultured Thermomicrobiabacterium (FN421261)

98

2 Thermotogae Mesotoga sp. VNs100 (HE818616) 1003 Firmicutes Uncultured Clostridiales

bacterium(HQ183782)

95

4 Firmicutes Syntrophomonas zehnderiOL-4 (NR_044008)

96

5 Firmicutes Uncultured Pelospora sp.(JX575841)

96

6 Firmicutes Uncultured Anaerovorax sp.(JN692194)

100

7 Thermotogae Thermotogae bacterium cloneL11_1_9 (JX473505)

100

8 Chloroflexi Uncultured Chloroflexi bacterium(DQ330295)

94

9 Chloroflexi Uncultured Anaerolinea sp.(EF613382)

99

10 a-Proteobacteria Uncultured proteobacterium(JN806324)

98

11 d-Proteobacteria Desulfatibacillum sp. cloneH12_1_12(JX473536)

100

12 a-Proteobacteria Erythrobacter citreus (EU888015) 100A Methanosaeta Methanosaeta concilii (AB679168) 99B Methanosaeta Uncultured Methanosaeta sp.

(GU475185)99

C Methanosaeta Uncultured Methanosaeta sp.(JX576101)

99

D Methanobacterium Methanobacterium kanagiense(AB368917)

99

E Methanobacterium Uncultured Methanobacterium sp.(EU888015)

99

F Methanobacterium Methanobacterium beijingense(AY552778)

98

G Methanobacterium Methanobacterium kanagiense(AB368917)

97

Y. Dang et al. / International Biodeterioration & Biodegradation 95 (2014) 76e8382

bacterium represented by band 1 was related to Thermomicrobiabacterium (FN421261), with a similarity of 98%. Thermomicrobiabacterium belongs to phylum Chloroflexi, which was frequentlyobserved in anaerobic bioreactor with strong ability to survive inmany kinds of wastewaters (Wu et al., 2001; Bj€ornsson et al., 2002).The bacterium represented by band 2 was related to Mesotoga sp.VNs100 (HE818616), with 100% similarity.Mesotoga belong phylumThermotogae, which preferred to living in the mesophilic and high-salt conditions. Conners et al. (2006) has reported that Thermotogaecan use a variety of complex hydrocarbons for free hydrogen pro-duction. The bacteria represented by band 3 and band 4 were allbelonged to phylum Firmicutes and class Clostridiales. The bacteriabelonging to Clostridiales, usually with spore-forming character-istic, canwithstand harsh external environment (Heider and Fuchs,1997). Therefore, this kind of bacteria could survive in high calciumconcentration and maintained the dominant position in sludge.

For archaea, all the detected archaea belonged to 2 genus,Methanosaeta and Methanobacterium. The archaea represented byband A and band B were the dominant archaea in the sludge duringthewhole research period. Theywere all belonged toMethanosaeta,which was a kind of acetoclastic methanogens and was always thedominant archaea in other anaerobic bioreactor (Liu et al., 2002;Hulshoff Pol et al., 2004; Rinc�on et al., 2006). The dominance ofacetoclastic methanogens guaranteed the COD removal efficiencyof EGSB reactor. The archaea represented by band A was related toMethanosaeta concilii (AB679168), with a similarity of 99%. It hasbeen frequently reported (Bainotti et al., 1997; Rinc�on et al., 2006)that Methanosaeta concilii plays an important role in the granules

formation, and can remove small organic acids such as acetic acidand acetate, contributing to the stable performance of the EGSBreactor. At the end of the operation in EGSB reactor (132 d) withinfluent calcium concentration of 7000 mg/L, the survival of mostarchaea were inhibited, and the growth and bioactivity of thedominant archaeawere affected by the high calcium concentration.Therefore, the metabolites of bacteria (e.g. VFAs) could not befurther utilized by methanogens transferring to methane, resultingin accumulation of VFAs. This is the main reason for the CODremoval decline.

5. Conclusions

Suppression threshold of calcium concentration and impact ofcalcium precipitation for the anaerobic granular sludge treatingfresh leachate from MSW incineration plant with extremely highcalcium concentration was investigated. High calcium concentra-tion lead to bioactivity reduction and calcium precipitation ingranules, resulting in the treatment efficiency declined. Comparedwith calcium precipitation, high calcium concentration was mainlyresponsible for the decrease of COD removal efficiency in treatmentprocess. The calcium suppression threshold for the sludge was5000 mg/L in both static tests and EGSB reactor operation. In thereal projects, calcium in influent should be controlled under thisvalue to ensure the treatment efficiency.

Acknowledgment

This work was supported by the Fundamental Research Fundsfor the Central Universities (BLYJ201401), Beijing Science andTechnology Plan Projects (Beijing Science and Technology Com-mission), as well as the National Natural Science Foundation ofChina (51278052).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibiod.2014.05.016.

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