Bioresource Technology 101 (2010) 2988–2995

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Bioresource Technology

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Comparison of nitrogen removal and microbial distribution in wastewatertreatment process under different electron donor conditions

Sora Park a, Jiyun Seon a, Imgyu Byun b, Sunja Cho c, Taejoo Park d, Taeho Lee a,*

a Department of Civil and Environmental Engineering, Pusan National University, Busan 609-735, Republic of Koreab Institute for Environmental Technology and Industry, Pusan National University, Busan 609-735, Republic of Koreac Division for Ubiquitous-Applied Construction of Port Logistics Infrastructures, Pusan National University, Busan 609-735, Republic of Koread Korea Environment Institute, Seoul 122-706, Republic of Korea

a r t i c l e i n f o

Article history:Received 28 August 2009Received in revised form 25 November 2009Accepted 5 December 2009Available online 6 January 2010

Keywords:Autotrophic denitrificationBiological nitrogen removalMicrobial communitySpent caustic

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.12.012

* Corresponding author. Tel.: +82 51 510 2465; faxE-mail address: leeth55@pusan.ac.kr (T. Lee).

a b s t r a c t

The applicability of modified spent caustic (MSC) as an electron donor for denitrification was evaluated ina lab-scale reactor for the Bardenpho process under various electron donor conditions: (A) no electrondonor, (B) methanol, (C) thiosulfate and (D) MSC conditions. TN removal efficiency varied in each condi-tion, 23.1%, 87.8%, 83.7% and 71.7%, respectively. The distribution ratio of nitrifying bacteria and DGGEprofile including sulfur-reducing or oxidizing bacteria also varied depending on the conditions. Theseresults indicated that the MSC would be used as an efficient electron donor for denitrification by auto-trophic denitrifier in wastewater treatment process.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Denitrification is a microbial process in which nitrate is con-verted into nitrogen (N2) gas. Biological denitrification is usuallydivided into heterotrophic denitrification and autotrophic denitri-fication depending on its electron donor source. Heterotrophicdenitrification is commonly coupled with nitrification for remov-ing nitrogen from domestic wastewater, and the heterotrophicdenitrifiers utilize organic matters such as ethanol and methanolas electron donors (Rittmann and McCarty, 2001). However, meth-anol is difficult to handle, and the residual methanol in the effluentcan pose a toxicity problem. As an alternative to heterotrophicdenitrification for biological wastewater treatment, autotrophicdenitrification has been drawing attention because it requires nocarbon source.

Autotrophic denitrification can be implemented using the re-duced sulfur compounds (H2S, S2O2�

3 , S4O2�6 , S2O2�

3 ), and the stoi-chiometric equations of autotrophic denitrification using sulfurcompounds are as follows:

1:1SþNO�3 þ0:4CO2þ0:76H2Oþ0:08HCO�3 þ0:08NHþ4! 0:08C5H7O2Nþ0:5N2þ1:1SO2�

4 þ1:28Hþ ð1Þ

ll rights reserved.

: +82 51 514 9574.

0:844S2O2�3 þNO�3 þ0:347COþ2 0:434H2Oþ0:086HCO�3

þ0:086NHþ4 ! 0:086C5H7O2Nþ0:5N2þ1:689SO2�4 þ0:697Hþ ð2Þ

0:421H2Sþ0:421HS� þNO�3 þ0:346CO2þ0:086HCO�3þ0:086NHþ4 ! 0:086C5H7O2Nþ0:5N2þ0:842SO2�

4

þ0:434H2Oþ0:262Hþ ð3Þ

while heterotrophic denitrification produces alkalinity, autotrophicdenitrification consumes alkalinity as shown in Eqs. (1-3). There-fore, adequate alkalinity source has to be supplied for sulfur-basedautotrophic denitrification.

In petrochemical plants such as naphtha cracking and ethylenereproduction, fresh caustic (NaOH) is used to remove the hydrogensulfide (H2S) gas which causes a corrosion problem. This solutionwhich absorbs H2S gas is generally referred to spent caustic (SC)(Rajganesh et al., 1995). SC is usually treated by wet air oxidation(WAO), fenton oxidation and incineration with auxiliary fuel; how-ever, these treatment methods are quite expensive and can causesecondary environmental pollution.

Since SC contains a high concentration of sulfide and alkalinityresulting from NaOH solution, it may serve as a sulfur and alkalin-ity source for autotrophic denitrification. When we applied SC asan electron donor for treating domestic wastewater in our previousstudy, (Park et al., 2008b) we came across some problems. One isthe presence of non-readily biodegradable organic matter such asbenzene, toluene, xylene, ethylbenzene and phenols, and the otheris the excessive alkalinity for autotrophic denitrification which

S. Park et al. / Bioresource Technology 101 (2010) 2988–2995 2989

causes high pH in the process (Sheu and Weng, 2001; (Park et al.,2008b).

The previous studies on the sulfur-based autotrophic denitrifi-cation have usually focused on the individual microbial species,especially Thiobacillus denitrificans which is the most well knownautotrophic denitrifier, and its physiology and kinetics (Claus andKutzner, 1985). We have extended the analysis of microbial com-munity for ammonia-oxidizing bacteria and nitrite-oxidizing bac-teria as well as T. denitrificans by using fluorescent in situhybridization (FISH) method (Park et al., 2008b). From the FISHresults, it was confirmed that the addition of SC affected on nitrify-ing bacteria and T. denitrificans. However, it was difficult to get en-ough information to understand variation of microbial structurecaused by adding SC. Therefore, the study using other molecularmethods is needed to better evaluate the autotrophic denitrifyingmicrobial community structure; this would be helpful to the pro-cess operation and maintenance.

The main objective of this study is to evaluate the applicabilityof modified spent caustic (MSC) from which hazardous and odor-ous substances have been removed for treating domestic wastewa-ter. We compared the pollutants (i.e., organic matters, nitrogen)removal performance of various electron donors such as methanoland thiosulfate. The variations in the relative distribution of nitri-fying bacteria were analyzed by using FISH, and the dynamics ofmicrobial community structure depending on the electron donorswas investigated through a DGGE.

2. Methods

2.1. Lab-scale Bardenpho process operation

To evaluate the process performances that depend on electrondonors for denitrification, a lab-scale reactor of the Bardenpho pro-cess was operated at room temperature. The reactor consists of an-oxic tank (1), aerobic tank (1), anoxic tank (2), aerobic tank (2) andclarifier, as illustrated in Fig. 1. The effective volume of the aerobictanks and anoxic tanks were 5.2 L, and the activated sludge fromSuyoung WWTP (Busan, Korea) was used for seeding. Nitrate andmixed liquor suspended solids (MLSS) were circulated by the inter-nal and external cycle, and the ratios of the internal and externalrecycle were fixed at 1.0 and 1.5, respectively. MSC and externalcarbon sources were injected into the anoxic tanks based on the to-tal influent nitrogen concentrations. A synthetic wastewater con-taining glucose 75 mg/L, NH4Cl 133.75 mg/L, KH2PO4 17.55 mg/L,NaHCO3 450 mg/L, KCl 17.5 mg/L, CaCl2�2H2O 17.5 mg/L, NaCl37.5 mg/L, MgSO4�7H2O 12.5 mg/L and trace element solution

Fig. 1. Schematic diagram of the lab-scale

(EDTA 0.5 g/L, FeSO4�7H2O 0.2 g/L, ZnSO4�7H2O 0.01 g/L,MnCl2�4H2O 0.003 g/L, H3BO3 0.03 g/L, CoCl2�6H2O 0.02 g/L,CuCl2�2H2O 0.01 g/L, NiCl2�6H2O 0.002 g/L, Na2MoO4�2H2O0.003 g/L) 1 mL/L was used as an influent. The characteristics ofinfluent and operating conditions are shown in Tables 1 and 2,respectively.

2.2. The preparation of modified spent caustics

In order to decompose volatile organic compounds such as ben-zene, toluene, ethylbenzene, xylene and odorous substances likemethylmercaptane and ethylmercaptane, ultrasonic wave was ap-plied (600 Watts/L for 1 h). The ultrasonic irradiation in SC wasconducted with the ultrasonic generator (VC750, Sonics & Materi-als, USA) (Lee et al., 2009).

Since SC contains an excessive alkalinity for sulphur-utilizingautotrophic denitrification, the SC injection to the process couldcause a rising of pH in the reactors. Therefore, sulphur and alkalin-ity concentration has to be balanced for preventing pH increase.There are two methods to balance sulfur and alkalinity concentra-tion. One is to control pH by addition of acids, and the other is toadd a sulfur source. In this study, the sulfur source (Na2S2O3�5H2O)was added to the spent caustics to balance sulphur and alkalinityconcentration, and this is termed as modified spent caustic(MSC). The composition of SC and MSC is compared in Table 3.

2.3. Fluorescent in situ hybridization

All in situ hybridizations were conducted using the procedureby Manz et al. (1994). The samples were fixed via immersionovernight in a freshly prepared paraformaldehyde solution (4% inphosphate buffered saline, PBS) at 4 �C. Thereafter, the sampleswere rinsed in PBS solution. Each sample was immobilized on agelatine-coated glass slide. The sample was finally dehydratedvia successive passage through an ethanol solution and dried.The fixed samples were hybridized first by sequentially spikingwith 8 lL of hybridization buffer (0.9 M NaCl, 20 mM Tris–HCl(pH 7.2), 0.01% sodium dodecyl sulphate (SDS)), formamide atthe concentrations shown in Table 4 and 2 lL of fluorescentprobes. The samples were then quickly transferred to a pre-warmed moisture chamber of 46 �C. Finally, the slides were dippedinto a washing solution at 48 �C. After hybridization, digital imagesof the aggregates were taken with a fluorescence microscope (ZeissAxioskop 2plus, Germany) and visualized with Zeiss Axiovisiondigital imaging software. Analyses were conducted with the

Bardenpho process used in this study.

Table 1Characteristics of the synthetic wastewater used in this study.

Item pH SCODCr (mg/L) NHþ4 –N (mg/L) TN (mg/L) Alkalinity (mg CaCO3/L)

Concentration 7.4–7.9 (7.5)a 55–74 (64) 24–36 (30) 24.3–39.9 (31.6) 262–330 (308)

a () is mean value during the operating days.

Table 2Operating conditions.

A B C D

Injected tank Anoxic 1 Anoxic 2 Anoxic 1 Anoxic 2 Anoxic 1 Anoxic 2 Anoxic 1 Anoxic 2MeOH dosage (mg COD/L influent) – – 123.0 52.5 – – – –

Thiosulfate dosage (mg S2O2�3 /L influent) – – – – 113.0 22.7

MSC dosage (mL MSC/L influent) – – – – – – 1.4 0.6COD/N ratio – – 3.7–4.3 2.7–8.9 – – – –

S2O2�3 /N ratio – – – – 3.3–3.9 1.05–22.7 1.03–1.25 0.7–6.0

S/N ratio – – – – – – 0.7–0.8 0.4–3.9HRT (h) 6 6 8 8

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standard software package using the Carl Zeiss Imaging Solutionsystem (Zeiss, Germany).

The following rRNA-targeted oligonucleotides were used:EUB338, Nso190, Ntspa662 and Nit3. The oligonucleotides werelabelled with fluorescein isothiocyanate (FITC) or with a hydro-philic sulfoindocyanine dye (CY3) at the 50 end by CoreBioSystem(Seoul, Korea). All probe sequences, hybridization conditions, andreferences are provided in Table 4.

2.4. DNA extraction and amplification

DNA was extracted from the sludge in anoxic reactors. Sampleswere centrifuged at 10,000g for 1 min and the supernatant was

Table 3The characteristics of spent caustic (SC) and modified spent caustic (MSC).

Items Spent caustic Modified spent caustic

pH 13.1–13.6 (13.4)a 13.0–13.4 (13.3)a

Alkalinity (mgCaCO3/L) 39,000–87,500 (66,800) 37,500–86,400 (65,200)SS (mg/L) 3100–10,360 (6691) 5–120 (30)TOC (mg/L) 1091–2266 (1858) 1120–2522 (1937)Benzene (mg/L) 6.23–20.81 (14.10) N.D.Toluene (mg/L) 0.13–0.48 (0.31) N.D.Ethylbenzene (mg/L) 0.00–0.30 (0.06) N.D.Xylene (mg/L) 0.01–0.22 (0.09) N.D.M-mercaptan (mg/L) 2.05–7.26 (4.03) N.D.E-mercaptan (mg/L) 0.23–0.63 (0.35) N.D.Phenols (mg/L) 1.8–33.8 (17.8) N.D.NHþ4 –N (mg/L) 80–426 (292) 30–340 (195)S2� (mg/L) 7020–17,983 (13,977) 6950–16,936 (13,653)

S2O2�3 (mg/L) N.D. 25,400

SO2�4 (mg/L) 2236–6240 (3944) 2446–8381 (4725)

N.D.: Not detected.a () is mean value.

Table 4Oligonucleotide probes used for FISH analysis in this study.

Probe Specificity Sequence(50–30)

EUB338 Most bacteria GCAGCCACCCGTANso190 Betaproteobactrial ammonia-oxidizing bacteria CGATCCCCTGCTTNit3 Nitrobacter spp. CCTGTGCTCCATGNtspa662 Genus Nitrospira GGAATTCCGCGCT

a 16S rRNA position according to Escherichia coli numbering.b Formamide concentration in the hybridization buffer.c Sodium chloride concentration in the washing buffer.

removed and resuspended in 1 mL of distilled water. PowerSoil™DNA kit (Mo Bio Labs, Carlsbad, CA) was used to extract DNA fol-lowing the manufacturer’s instructions. The electrophoretic profileof the DNA products was determined using a 1% agarose gel. Theextracted DNA was amplified using a Mastercycler gradient auto-mated thermal cycler (Bio-Rad Laboratories, Inc., Hercules, USA)with targeted 16S rDNA at the following program: an initial dena-turation at 95 �C for 2 min; 29 cycles of denaturation (20 s at95 �C), annealing (40 s at 55 �C), extension (30 s at 72 �C); and afinal extension at 72 �C for 5 min. For amplification of the 16SrDNA, the forward primer 10F (50-AGAGTTTGATCMTGGCTCAG-30)and the reverse primer 1392R (50-ACGGGCGGTGTGTACAAG-30)were used. After that, the forward primer 341F (50-CCTACGG-GAGGCAGCAG-30) was used with a GC clamp (50-CGCCGCGCGGC-GGGCCCCCGCGGGGC-30). The reactions were carried out in a25 lL volume containing 1 lL of template DNA, 0.25 lL of the for-ward and reverse primers (10 pmol), 2.5 lL of 10x Taq buffer,10 lL of 10 mM dNTP and 0.125 lL of DNA polymerase (Solgent,Daejeon, Korea). PCR products were checked electrophoreticallyand were purified using a PCR purification kit (Bioneer, Alameda,CA).

2.5. DGGE, sequence analysis of bands from DGGE gel

DGGE was performed using a Dcode system (Bio-Rad, Hercules,CA). Samples were loaded onto a 6% polyacrylamide gel in 0.5x TAEbuffer (40 mM Tris–acetate, 1 mM EDTA, pH 8.3). A denaturantgradient ranging from 30% to 60% denaturant (100% denaturantconstituted 7 M urea and 40% v/v formamide) was used. Gelswere run at 60 �C for 8 h at a constant voltage of 200 V, and thenstained for 30 min in ethidium bromide (Bio-Rad). The band profilewas identified using the ultraviolet transilluminator (Uvitec,Cambridge, UK). DGGE bands chosen for sequence analysis were

Target sizea FA (%)b NaCl (mM)c Reference

GGTGT 338–355 0–50 - Amann et al., 1990TTCTCC 190–208 55 0.020 Mobarry et al., 1996CTCCG 1035–1048 40 0.056 Wagner et al., 1996CCTCT 662–679 35 0.079 Daims et al., 2001

S. Park et al. / Bioresource Technology 101 (2010) 2988–2995 2991

excised and transferred to 30 lL TE buffer. To resuspend DNA, eachsample was heated at 60 �C and frozen at �80 �C three times for15 min. The DNA was reamplified using the same primers. Afterpurification, PCR products were sequenced using an ABI 3730XLcapillary DNA sequencer (Applied Biosystems, Franklin Lakes, NJ)and an ABI Prism Bigdye terminator cycle sequencing ready reac-tion kit version 3.1 (Applied Biosystems).

2.6. Statistical analysis

Pearson correlation coefficient was analyzed to determine therelationships between microbial community and electron donors.Statistical analyses were performed using the Statistical Packagefor Social Sciences, fingerprinting II informatix software version3.0 for windows (Bio-Rad Laboratories, Inc., Hercules, USA).

2.7. Analytical methods

NO�3 –N, NO�2 –N, and SO2�4 concentrations were determined

using ion chromatography (DX-300, DIONEX, Sunnyvale, USA).The soluble chemical oxygen demand (SCOD) and NHþ4 –N concen-trations were determined using an auto analyzer (AA3, Bran + -Luebbe, Germany) after the filtration of the sample through a0.45 lm membrane filter. Phenols and BTEX concentrations weremeasured via gas chromatography mass spectrometry (HP5973 N, USA). The pH and dissolved oxygen (DO) concentrationswere measured using an Orion Research pH meter (230A, ThermoFisher Scientific Inc., Walthan, USA) and a DO meter (YSI 58, Fon-driest Environmental, Inc., Dayton, USA), respectively. The sulfurcontent was then measured via inductively-coupled plasma atomicemission spectrophotometry (JY ULTIMA 2C, Thermo Jarrell Ash,Michigan, USA), and the alkalinity and suspended solids were as-sessed via Standard Methods (APHA and WA, 1998).

3. Results and discussion

3.1. Organic matter removal

The SCODCr concentration in the influent and effluent in eachcondition is presented in Fig. 2. The SCODCr removal efficiencywas the highest in condition A when no external electron donorwas supplied. The average SCODCr removal efficiency was 75.8%,70.7%, 72.2% and 61.9%, in conditions A, B, C and D, respectively.We infer that most of the removed organic matter was used to het-

Operating time (day)0 20 40 60 80 100

CO

D c

once

ntra

tion

(mg/

L)

0

20

40

60

80

A B C D

Fig. 2. SCODCr concentrations of influent (d) and effluent (s) in different electrondonor conditions; no electron donor (A), methanol (B), thiosulfate (C) and MSC (D).

erotrophic denitrification in condition A because the COD/N ratioof the influent is low. Methanol was injected for additional hetero-trophic denitrification in condition B. The methanol dosage wascalculated on the basis of the TN concentration of the influent asdescribed in Table 2. The TN removal efficiency did not reach100%. Therefore, the residual methanol that could not be used forheterotrophic denitrification was discharged and reflected as an in-crease in the organic matter concentration in the effluent. The con-dition C in which thiosulfate was supplied, the SCODCr removalefficiency was similar to condition A. In condition D, the SCODCr

increment in the effluent was observed because MSC was injectedbased on S/N ratio without considering the organic matters in MSC.We expected that the MSC dosage can be reduced if the organicmatter concentration of MSC is calculated to be applied in waste-water treatment. Since MSC contains organic matter, the applica-tion of MSC to the BNR process can bring about heterotrophicdenitrification as well as autotrophic denitrification.

3.2. Nitrification and nitrifying bacteria

To maintain a high TN removal efficiency, complete nitrificationis necessary. While the lab-scale Bardenpho process was in opera-tion, the nitrification efficiency was over 98.3%. The NHþ4 –N con-centration is displayed in Fig. 3a, and the nitrification efficiencywas stable in accordance with the various electron donor supplies.In the nitrification step, the nitrifying bacteria convert ammonium(NHþ4 ) to nitrate (NO�3 ), consisting of ammonia-oxidizing bacteria(AOB) and nitrite-oxidizing bacteria (NOB). We investigated therelative distribution ratio of AOB and NOB depending on the vari-ous electron donors. The relative population of nitrifying bacteriaof aerobic tank (1) in each condition is presented in Fig. 3b. The rel-ative distribution ratio of AOB in aerobic tank (1) was 8.4%, 8.2%,6.7% and 6.5% in conditions A, B, C and D, respectively. As the sub-strate uptake rate is connected with growth rate directly, AOBchanged accordance with the nitrogen loading rate in condition Cand D. Compared with condition A, the nitrogen loading rate de-creased in condition C and D, 20.8% and 22.3%, respectively, be-cause of the increase in HRT. The relative distribution ratio ofrepresentative NOB, Nitrobacter spp., were 6.6%, 8.5%, 6.2% and6.1% and in the case of Nitrospira genus, the distribution ratiowas 10.4%, 8.8%, 7.8% and 7.3%, respectively.

The relative distribution ratio of nitrifying bacteria (the sum ofAOB, Nitrobacter spp. and Nitrospira genus) ranged from 19.9% to25.4%. It is generally known that the optimal pH for Nitrobacterspp. was the range of 7.2–7.6 (Villaverde et al., 1997). In this study,the decrease of Nitrobacter spp. was caused by pH variation. The pHof aerobic tank (1) in each condition was 7.47, 7.58, 7.64 and 7.71,respectively. In the nitrifying biofilm reactor with the organic car-bon, the Nitrospira genus was affiliated and Nitrobacter sp. was onlydetected restrictively (Nogueira et al., 2002). In this study, how-ever, both of them showed a similar distribution ratio.

Although it has been reported that nitrifying bacteria is sensi-tive to pH, temperature, free ammonia, carbon disulfide and othercompounds (Bundy and Bremner, 1973; Bedard and Knowles,1989; Park et al., 2008a), all electron donors tested in this study,including MSC even, did not give any inhibitory effect onnitrification.

3.3. TN removal and autotrophic denitrification

TN removal efficiency was 23.1% in condition A in which onlythe heterotrophic denitrification occurred. The TN concentrationin each condition is shown in Fig. 4a. In condition B, due to themethanol injection as an external carbon source, the TN removalefficiency increased significantly to 87.8%. In conditions C and D,which simultaneous heterotrophic and autotrophic denitrification

Fig. 3. Influent (d) and effluent (s) concentrations of NHþ4 –N (a) and the relative distribution ratio of nitrifying bacteria in aerobic tank (1) (j) AOB, (h) Nitrobacter spp., ( )Nitrospira genus, (d) pH, (b) under different electron donor condition; no electron donor (A), methanol (B), thiosulfate (C) and MSC (D).

Fig. 4. Influent (d) and effluent (s) concentration of total nitrogen (a), and the efficiencies of autotrophic (j), heterotrophic (h) and total ( ) denitrification (b) underdifferent electron donor condition; no electron donor (A), methanol (B), thiosulfate (C) and MSC (D).

2992 S. Park et al. / Bioresource Technology 101 (2010) 2988–2995

are expected to occur, the TN removal efficiency was 83.7% and71.7%, respectively. The lower TN removal efficiency comparedwith condition B is attributed to the fact that autotrophic denitri-fication can be implemented by autotrophs which have a lowergrowth rate (Tchobanoglous et al., 2003).

Furthermore, the autotrophs using sulfur can carry out theautotrophic denitrification perfectly under the pH between 7.5and 8.0 and temperature of 30 �C (Claus and Kutzner, 1985). Inall of the conditions, the pH was nearly close to the optimum pHrange. The pH value of anoxic tank (1) was 7.21, 7.43, 7.42 and7.48 in each condition, and that of anoxic tank (2) was 7.22, 7.33,7.46 and 7.33, respectively. The MSC injection did not cause thesignificant pH increase of the anoxic tanks, indicating that theaddition of sulfur source made more alkalinity consumed. Theeffluent pH was 7.47, 7.55, 7.68 and 7.59 in each condition. Onthe other hand, the operating temperature of the reactors was low-er (10.9–18.2 �C) than optimum value. Accordingly, it seemed thatthe lower TN removal efficiency in condition C and D would be

caused by the lower temperature. Under condition A in which onlyheterotrophic denitrification was conducted, 6.5 mg SCODCr wasremoved as 1 mg of nitrate–nitrogen was denitrified. The TN re-moval efficiency by autotrophic denitrification in each conditionwas calculated using the following equation (Park et al., 2008b);

TRauto ¼ TRtotal � TRhetero ð4Þ

where TRauto = the TN removal efficiency by autotrophic denitrifica-tion, TRtotal = the TN removal efficiency, and TRhetero = the TN re-moval efficiency by heterotrophic denitrification.

Although MSC was supplied as an electron donor for autotroph-ic denitrification, MSC contains organic matters as shown in Table3. The mean organic matter in MSC for heterotrophic denitrifica-tion was about 1937 mg of TOC or 5165 mg of COD/L. Therefore,the organic matters in the influent and MSC were considered incalculating the TN removal efficiency by heterotrophic denitrifica-tion. However, the increase in organic matter caused by the MSC

S. Park et al. / Bioresource Technology 101 (2010) 2988–2995 2993

injection was negligible because a small quantity of MSC (2 mL/Linfluent) was injected. Fig. 4b shows the TN removal efficiencyby autotrophic and heterotrophic denitrification.

In conditions A and B, no autotrophic denitrification was imple-mented since sulfur source was not provided. The autotrophicdenitrification efficiency was 59.3% and 50.0%, respectively, in con-ditions C and D.

3.4. Sulfate and sludge production

In the process of sulfur-utilizing autotrophic denitrification, sul-fate is one of the end products, and the amount of produced sul-fate/removed nitrate showed various values depending on theform of sulfur source, as represented in Eqs. (1-3). The sulfate con-centration in each condition is shown in Fig. 5. The sulfate concen-tration values ranged from 237.5 to 275.0 mg/L after injectingthiosulfate or MSC, while they ranged from 83.6 to 103.3 mg/Lwhen they were caused by the sulfate concentration of the influentbefore the injection of sulfur source. We think that these levels ofproduced sulfate do not give any harmful effect on the environ-ment, because the treated wastewater is directly discharged to

Fig. 5. Sulfate concentrations in effluent under different electron donor conditions;no electron donor (A), methanol (B), thiosulfate (C) and MSC (D).

Fig. 6. Dendrogram based on Pearson correlation coefficient among DGGE band profile ofand the applied electron donors are as follows; (a) no external donor, (b) methanol, (c)

the sea that contains a natural sulfate concentration of about2700 mg/L (Park et al., 2009).

Dissolved organic pollutants in domestic wastewater transformto biomass, carbon dioxide and water through biological processes.Therefore, the main by-product is sewage sludge, which consists ofmicrobial biomass (Rocher et al., 1999). The amount of sewagesludge produced in Korea was about 7630 ton/day (2007). Its han-dling and disposal is an issue of particular concern not only be-cause the cost of sludge treatment is high (Yoshida et al., 2009)but also because dumping sewage sludge into the sea will be pro-hibited in Korea from 2012 in accordance with the London Conven-tion. One of the merits of autotrophic denitrification is the lowersludge production than the heterotrophic denitrification (Clausand Kutzner, 1985).

The sludge yields were 0.38, 0.21 and 0.28 g VSS/g NO�3 –N in B,C and D conditions, respectively. The value of condition B was low-er than the values reported in the literature (Engberg and Schroe-der, 1975; Timmermans and Van Haute, 1983), and the yield wasreported to be 0.31 mg MLVSS/mg NO�3 through the batch cultiva-tion of microorganism using SC (Byun et al., 2008). This study hasshown clearly that the sludge production under autotrophic deni-trification is lower than that under heterotrophic denitrification.

3.5. DGGE analysis of 16S rDNA fragments

DGGE was conducted to compare the changes in the structureand diversity of the microbial community under different electrondonor conditions. DGGE band profile and Pearson correlation coef-ficient are represented in Fig. 6. The band patterns of samples col-lected from the different electron donor conditions were quitedifferent from each other, suggesting that the structure and diver-sity of the microbial community in each condition would be differ-ent. The similarity between anoxic tank (1) and (2) was over 90.8%according to the Pearson coefficient because the process was recir-culation-type system.

To investigate the effect of electron donors on microbial com-munity, representative bands were extracted from the gel and se-quenced. The results are shown in Table 5. Band 2 showed 97%similarity to Trichococcus flocculiformis, which is known as one ofthe filamentous bacteria responsible for bulking in activated

16S rDNA fragment from the different electron donor conditions; S/S: seeded sludgethiosulfate and (d) modified spent caustic.

Table 5Taxonomic identification of representative phylotypes in the DGGE profiles shown inFig. 5.

Bandno.

Most closest strain Similarity(%)

Accession no. of the relatedstrain in Genbank

1 Arcobacter sp. R-28314 97 AM0841142 Trichococcus

flocculiformis97 EU434527

3 Uncultured epsilonproteobacterium

99 FJ535178

4 Uncultured epsilonproteobacterium

100 FJ967862

5 Bacillus sp. Nu-2 98 FJ9843326 Uncultured

rubrobacteridaebacterium

93 AM935542

7 Uncultured bacterium 92 DQ6617478 Uncultured bacterium 88 EU3328009 Uncultured bacterium 87 FJ70583610 Uncultured soil

bacterium97 EU861901

11 Uncultured bacterium 100 CU91850512 Desulfotomaculum sp.

DEM-KMe99-386 AJ276566

13 Uncultured epsilonproteobacterium

100 FJ967860

14 Uncultured bacterium 100 DQ988298

2994 S. Park et al. / Bioresource Technology 101 (2010) 2988–2995

sludge plants (Scheff et al., 1984), and it was detected only in theseeding sludge. After injecting electron donors for denitrification,bands 3 and 4 appeared. They showed a high similarity to uncul-tured epsilon proteobacteria isolated from an anaerobic digesterwhich treats municipal wastewater sludge and sulfur-rich waterfrom sulfurian sedimentary rock. Bands 6 and 7 appeared withthe injection of the sulfur source, and band 6 was found to beuncultured rubrobacteridae bacterium isolated from the pilot-scalebioremediation process of a hydrocarbon-contaminated soil. About7 bands (band 5, 8, 9, 11, 12, 13 and 14) appeared after MSC wasinjected into the anoxic tanks. Band 5 was identified as Bacillussp. Nu-2 isolated from wastewater in Korea, and band 8 is closelyrelated to uncultured bacteria which are originated from an aero-bic enhanced biological phosphorus removal sequencing batchreactor. Band 9 was related to uncultured bacteria which are orig-inated from soil contaminated with herbicide and dioxin. Band 11was closely associated with uncultured bacterium isolated fromthe anaerobic digester which treats municipal wastewater sludge.

Bands 12 and 13 were considered to be related with sulfur com-pounds. Band 12 was confirmed as Desulfotomaculum sp. DEM-KMe99-3 (Stubner and Meuser, 2000), which is one of the sulfatereducing bacteria (SRB) (Luptakova and Kusnierova, 2005). Desulfo-tomaculum has an ability to reduce sulfate to sulfide. Although sul-fate reduction is not considered as a main mechanism in anoxictanks, the presence of SRB is acceptable because sulfate was pro-duced via autotrophic denitrification using MSC. However, thisfunction of SRB was found to be slight in this study. Band 13 wasclosely linked to uncultured epsilon proteobacterium isolated fromsulfur-rich spring. The numerous reduced forms of sulfur com-pounds such as sulfide (H2S, HS�, S2�), elemental sulfur (S), thiosul-fate (S2O2�

3 ) are within the sulfurous springs (Orr, 1974). Thesefinding led us to assume that the uncultured epsilon proteobacte-rium detected in D condition utilized these forms of sulfur sourcesand denitrified autotrophically. Three bands (bands 3, 4 and 13)showed a high similarity to the uncultured epsilon proteobacteria.It has been reported previously that Thiomicrospira denitrificans,which can reduce nitrate while oxidizing sulfur source (sulfide/thiosulfate), is epsilon proteobacteria (Timmer-ten Hoor, 1975).

In addition, an uncultured soil bacterium (band 10) isolatedfrom nitrogen amended dry meadow surface soil (Nemergut

et al., 2008) and another uncultured bacterium (band 14) isolatedfrom membrane bioreactor were detected.

4. Conclusions

MSC was proven to be a comparable electron donor to methanoland thiosulfate, as MSC addition induced about 47.3% of enhancednitrogen removal. The application of different type of electron do-nors such as methanol, thiosulfate and MSC affected not nitrifica-tion efficiency, but AOB and NOB distribution. More preciseanalysis of microbial community structure by using DGGE of bac-terial 16s rDNA fragments suggested that sulfur-oxidizing bacteria,belonging to e-proteobacteria, seemed to utilize the sulfur sourcesfor autotrophic denitrification. Consequently, MSC would be usedas an effective electron donor for enhanced denitrification by auto-trophic denitrifying bacteria in wastewater treatment process.

Acknowledgements

This work was financially supported by Korea Ministry of Envi-ronment (MOE) as ‘Human resource development Project forWaste to Energy’, and the Core Environmental Technology Devel-opment Project for the Next Generation Program (Eco-Techno-phia-21), Korea Institute of Environmental Science & Technology,Republic of Korea.

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