Anammox for Nitrogen Removal From Anaerobically Pre-treated

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Anammox for nitrogen removal from anaerobically pre-treatedmunicipal wastewater: Effect of COD/N ratios on process performanceand bacterial community structure

Cntia Dutra Leal, Alyne Duarte Pereira, Fernando Terra Nunes, Lusa Ornelas Ferreira,Aline Carolina Cirilo Coelho, Sarah Kinaip Bicalho, Erika F. AbreuMac Conell, Thiago Bressani Ribeiro,Carlos Augusto de Lemos Chernicharo, Juliana Calbria de Arajo

Department of Sanitary and Environmental Engineering of Federal University of Minas Gerais (UFMG), Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil

h i g h l i g h t s

Anammox metabolism was inhibited by the addition of 487.5 mgL1 COD.A COD/N ratio of 5.0 (COD of 300 mg L1) did not affect nitrogen removal.Anaerobic effluent changed bacterial community structure and diversity.Anammox and denitrifying bacteria were both found in the reactor.Anammox can be applied to remove nitrogen from pre-treated municipal wastewater.

a r t i c l e i n f o

Article history:

Received 20 January 2016

Received in revised form 18 March 2016Accepted 19 March 2016

Available online 22 March 2016

Keywords:

Anammox

COD/N ratio

Anaerobic effluent

DGGE

Nitrogen removal

a b s t r a c t

Long-term effects of COD/N ratios on the nitrogen removal performance and bacterial community of an

anammox reactor were evaluated by adding a synthetic medium (with glucose) and real anaerobic efflu-

ent to a SBR. At a COD/N ratio of 2.8 (COD, 390 mgL

1) ammonium removal efficiency was 66%, whilenitrite removal remained high (99%). However, at a COD/N ratio of 5.0 (COD, 300 mg L1), ammonium

and nitrite removal efficiencies were high (84% and 99%, respectively). High COD, nitrite, and ammonium

removal efficiencies (80%, 90% and 95%, respectively) were obtained on adding anaerobically pre-treatedmunicipal wastewater (with nitrite) to the reactor. DGGE revealed that the addition of anaerobic effluentchanged the bacterial community structure and selected for DNA sequences related to Brocadia sinicaandChloroflexi. Adding glucose and anaerobic effluent increased denitrifiers concentration threefold. Thus,

the possibility of using the anammox process to remove nitrogen from anaerobically pre-treated munic-ipal wastewater was demonstrated.

2016 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic ammonium oxidation (anammox) is a microbial pro-cess that can be used, in principle, for the treatment of ammonium-rich wastewaters with a low C:N ratio. Anammox bacteria oxidize

ammonium (NH4+

) into nitrogen gas (N2) under anoxic conditionsusing nitrite (NO2) as the terminal electron acceptor (van de

Graaf et al., 1996; Strous et al., 1998). These are chemolitoau-totrophic bacteria characterized by slow growth (Strous et al.,1998; Kartal et al., 2011), belonging to the phylum Planctomycetes,

order Brocadiales.The anammox process simultaneously removes two pollutants,

ammonium and nitrite, converting them into gaseous nitrogen(Zhang et al., 2008). Inorganic carbon sources such as CO2 andHCO3

are particularly important for the cultivation of anammox

bacteria and favor the growth and activity of these microorganisms(van de Graaf et al., 1996). On the other hand, organic compounds

http://dx.doi.org/10.1016/j.biortech.2016.03.107

0960-8524/2016 Elsevier Ltd. All rights reserved.

Abbreviations: Anammox, anaerobic ammonium oxidation; BOD, biochemical

oxygen demand; COD, chemical oxygen demand; DGGE, denaturing gradient gel

electrophoresis; DO, dissolved oxygen; FISH, fluorescent in situ hybridization; TNK,

total nitrogen Kjeldahl; SBR, sequencing batch reactor; TSS, total suspended solids;

UASB, upflow anaerobic sludge blanket reactor; VSS, volatile suspended solid. Corresponding author.

E-mail address: [email protected](J.C. de Arajo).

Bioresource Technology 211 (2016) 257266

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(present in almost all types of wastewaters) may give rise to

adverse effects on anammox bacteria and may impair the anam-mox activity, especially at high concentrations (Gven et al.,2005; Chanchoi et al., 2008; Molinuevo et al., 2009). Inhibition ofthe anammox process by organic compounds may occur due toenzyme inactivation, and may be irreversible, resulting in cell

death (Gven et al., 2005). Another proposed mechanism to

explain anammox impairment is the competition between anam-mox bacteria and denitrifying heterotrophic bacteria for the elec-tron acceptor, nitrite. Since heterotrophic bacteria are able to

grow faster than autotrophic ones, they outcompete the anammoxbacteria and hinder their chemical reactions (Gven et al., 2005;Chanchoi et al., 2008; Lackner et al., 2008; Molinuevo et al., 2009 ).

There is no consensus in the literature on what is the organic

matter concentration (expressed as COD) and the COD to N ratiothat inhibits/or affects the anammox process. According toChanchoi et al. (2008) COD concentrations above 300 mgL1

(COD/N ratio of 2) may fully inhibit anammox reaction and con-comitantly favor the activity of denitrifying bacteria. Ni et al.(2012)verified that at COD/N ratios above 4 the anammox processwas impaired. Snchez-Guilln et al. (2014) investigating the

short-term effects of organic carbon addition (acetate and starch)verified that the COD/N ratios (2 and 6) applied had no significantinfluence on the anammox process.

Jenni et al. (2014) determined the influence of different COD

concentrations and hydraulic retention times on the nitrogenremoval (by the anammox process). The COD/N ratio was graduallyincreased up to 1.4 g COD g N1, while the hydraulic retention timewas reduced. After adding acetate and glucose (as carbon sources),

the efficiency reached nearly 95%. The results showed that with aratio of 0.8 g COD g N1 and higher there was a reduction in theabundance of anammox bacteria (estimated by FISH), but the num-ber ofCandidatus Brocadia fulgida cells increased. The authors also

found that alternating between acetate and glucose had no nega-tive effect on the community profile, and that the prevalence of

Ca. Brocadia fulgidacould be explained by the fact that this species

can oxidize acetate.Despite the previous findings, these studies did not address

ammonium or nitrogen removal from real anaerobically pre-treated municipal wastewater by the anammox process. In addi-tion, no study has investigated the long-term effects of adding

COD or increasing COD/N ratios (up to 5.0) in an anammox reactor.The anammox process has been successfully applied to remove

nitrogen at many wastewater treatment plants around the world,especially ammonium-rich wastewaters with low C:N ratios. Nev-

ertheless, few studies have investigated the potential application ofthe anammox process to remove nitrogen from effluents with lowammonium concentrations and high COD/N ratios, such as the

effluent from an upflow anaerobic sludge bed reactor (UASB) treat-ing domestic wastewater. Therefore, the objectives of this study

were: to evaluate the efficiency of an anammox reactor in remov-ing nitrogen when subjected to different COD/N ratios, and to eval-

uate the possibility of applying the anammox process to nitrogenremoval from anaerobically pre-treated municipal wastewater.Moreover, the bacterial community structure in the anammoxreactor during experiments with synthetic medium (before and

after glucose addition) and with real anaerobic effluent was inves-tigated and compared.

2. Materials and methods

2.1. Experimental set-up

A 2.0-L glass reactor (Benchtop Fermentor & Bioreactor BioFlo

/CelliGen 115, New Brunswick Scientific Co., Enfield, CT, USA) was

used for cultivation of anammox biomass in this study. The reactorwas monitored for 558 days and was operated in sequencing batch

mode with two daily cycles, one of 7 h (short cycle) and the otherof 17 h (long cycle), resulting in a hydraulic retention time (HRT) of24 h and total biomass retention. The SBR was fed with autotrophicmedium (van de Graaf et al., 1996; Dapena-Mora et al., 2004). The

concentrations of ammonium and nitrite in the medium ranged

from, respectively, 100 mg

L

1

to 150 mg

L

1

and 150 mg

L

1

to200 mgL1 (while the nitrite to ammonium ratio applied was keptaround 1.31.4), for 323 days prior to the phase in which the reac-

tor was exposed to different COD/N ratios.The temperature inside the reactor was kept at 35 C and pH

was adjusted to 7.5. Influent and effluent samples were collectedto monitor the concentrations of ammonium, nitrite and the

COD. Analyses of these parameters were performed according tothe Standard Methods for the Examination of Water and Wastew-ater (APHA, 2012). Biomass samples were taken from the reactor at

the end of each operational phase in order to investigate the bacte-rial diversity inside the reactor.

2.2. Anammox biomass

The SBR was inoculated with biomass taken from two bioreac-tors that showed anammox activity, both seeded with sludge frommunicipal wastewater treatment plants (detailed information of

these two bioreactors is presented in Pereira et al., 2014, andCosta et al., 2014). The microbial communities of the two inocula,characterized by 454 pyrosequencing, showed that the prevailingphyla were Proteobacteria, Firmicutes, Chloroflexi, Verrucomicrobia,

and Planctomycetes(Pereira et al., 2014; Costa et al., 2014).

2.3. Experiment applying different COD/N ratios to the SBR

The enrichment and cultivation period (Phase I) lasted323 days. In the following period (Phase II), different COD/N ratios(using synthetic medium with glucose) were applied to the SBR, by

gradually increasing the COD concentration. Glucose was used as acarbon source because it is a non-toxic organic compound and alsobecause anammox bacteria cannot degrade this substrate (Gvenet al., 2005; Jenni et al., 2014). Glucose was introduced into theSBR during the long cycle (17 h) through a septum located at the

top of the reactor using 50-mL syringes. Varying amounts of a con-centrated glucose solution were added to the reactor in order toachieve the desired final concentrations of 95, 195, 390, 487.5,and 300 mgL1, which corresponded to the COD/N ratios being

tested, i.e., 0.7, 1.4, 2.8, 3.5 and 5.0, respectively. In order to achievethese ratios, the concentrations of ammonium and nitrite in themedium were reduced to, respectively, 60 mgL1 and 80 mgL1

(while the nitrite to ammonium ratio applied was 1.33, same ratioused in Phase I).

The results obtained in Phase I were considered referenceresults for nitrogen removal by the anammox process in the SBR.

With a COD/N ratio of 3.5, a decrease in anammox activity wasobserved, and, for that reason, glucose feeding was stopped to pre-vent irreversible inhibition of anammox bacteria activity. The reac-tor was held in a recovery phase for 35 days, being fed only with

synthetic medium, while the concentrations of nitrite and ammo-nium were reduced to 30 mgL1 and 30 mgL1 respectively, and,after the recovery period, the test was performed with a COD/Nratio of 5.0 (COD of 300 mgL1 and nitrogen concentration of

60 mgL1). Statistical analysis (using KruskalWallis test) was per-formed to assess whether different concentrations of glucoseadded to the SBR altered the nitrite and ammonium removal effi-ciency, i.e., to verify if there would be statistical differences

between the reference values (median values of ammonium andnitrite removal efficiencies observed in Phase I-without the

258 C.D. Leal et al. / Bioresource Technology 211 (2016) 257266


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addition of glucose) and those values found in Phase II (in which

glucose was added to the reactor). KruskalWallis test (a= 5%)was performed, followed by a multiple comparisons test of medi-ans (a= 5%), using Statistica 8 software.

2.4. Experiment applying real anaerobic effluent to the SBR

In Phase III, the anammox reactor was fed with anaerobicallypre-treated municipal wastewater. A small parcel of municipalwastewater (taken from Arrudas WWTP the main plant of Belo

Horizonte city, Minas Gerais, Brazil) was pre-treated in a demo-scale UASB reactor (V= 16,800 L; average HRT = 8.6 h). The chemi-cal composition of the anaerobically pre-treated municipal

wastewater (real anaerobic effluent) is shown in Table 1. Theanaerobic effluent feeding process comprised two steps. In the first(Phase IIIA), 60% anaerobic effluent was added to 40% autotrophicmedium, containing 50 mgL1 of nitrite and 30 mgL1 of ammo-

nium. In the second (Phase IIIB), only anaerobic effluent supple-mented with 120 mgL1 of nitrite was added to the reactor,under the same operational conditions.

Statistical analysis (using KruskalWallis test) were performed

to assess whether the addition of anaerobic effluent to the SBRaltered the nitrite and ammonium removal efficiency, i.e., to verifyif there would be statistical differences between the reference val-ues (observed in Phase I) and those values found in Phase III. Krus-

kalWallis test (a= 5%) was performed, followed by a multiplecomparisons test of medians (a= 5%), using Statistica 8 software.Efficiencies in Phase III, in which anaerobic effluent (amended withnitrite) was added to the reactor, were compared to the previous

periods (Phase II, with glucose addition; and Phase I, without glu-cose addition).

2.5. Characterization of bacterial community by polymerase chain

reaction-denaturing gradient gel electrophoresis (PCR-DGGE)

Biomass samples were taken from the SBR at day 349 (Phase I,the control phase used as a reference for the anammox process),

day 448 (after the recovery step without glucose), day 482 (afterPhase II addition of glucose for different COD/N ratios), day 558(after Phase III addition of real anaerobic effluent) and representeach of the different phases of SBR operation. Genomic DNA was

extracted from 0.5 g of each sample using a PowerSoil DNA Isola-tion Kit (MO BIO Laboratories, USA). PCR was performed usingthe primer set 1055F (ATGGCTGTCGTCAGCT) and 1392R

(ACGGGCGGTGTGTAC) with a GC clamp, as described previously

(Ferris et al., 1996). PCR products were subjected to DGGE. DGGEwas performed with a Bio-Rad DCode Universal Mutation Detec-

tion System (Hercules, CA, USA) using an 8% polyacrylamide gelwith 5075% denaturing gradient for 16.5 h at 75 volts. Gels werestained with SybrGold (LifeTechnologies) and analyzed with theBioNumerics 7.1 software (Applied Maths, Austin, TX, USA). Band

profiles were compared using Dice similarity coefficient and the

dendrogram was generated with UPGMA method, with 1% positiontolerance.DNA gel bands were excised and re-amplified. The PCR products

were sent to Macrogen, Inc. (Seoul, Korea) for purification and uni-directional sequencing in a 3730XL Sequencer. These sequenceswere compared with sequences in the Ribosomal Database Projectby RDP Classifier (https://rdp.cme.msu.edu/classifier/classifier.jsp),

reaching a reliability level of 80%, and with sequences from theNational Center for Biotechnology Information database using theBasic Local Alignment Search Tool (Altschul et al., 1990).

2.6. Quantitative PCR

The abundance of anammox bacteria, denitrifiers, and total bac-

teria were investigated by real time quantitative PCR (qPCR) usingSybrGreen assays on biomass samples taken from the SBR at theend of Phases I, II and III (at day 349, 482 and 558, respectively).qPCR assays were conducted on a real-time PCR thermal cycler

(Applied Biosystems 7500 instrument). The total bacterial abun-dance was quantified using eubacterial 16S rRNA-targeted primers1055F (ATGGCTGTCGTCAGCT) and 1392R (ACGGGCGGTGTGTAC)(Ferris et al., 1996), and anammox bacteria were quantified using

primers Pla46F (GGATTAGGCATGCAAGTC) and Amx667R (ACCA-GAAGTTCCACTCTC). The abundance of denitrifiers was quantifiedusing primers targeting the nitrous oxide reductase gene nosZF

(CGYTGTTCMTCGACAGCCAG) and nosZ1622R (CGSACCTTSTTGCC-STYGCG) (Enwall et al., 2005). Each 20 lL reaction mixture con-tained 10 ng of template DNA, 375 nM forward and reverseprimers (each), and 10 lL of MaximaTM SybrGreen/ROX qPCR Mas-

ter Mix 2(Thermo Scientific, USA). Standard curves for qPCR weregenerated by 10-fold serial dilutions of plasmid DNA containingspecific target gene inserts.

3. Results and discussion

3.1. Nitrogen removal during operational Phases I, II and III

The performance of the SBR over 558 days is shown in Fig. 1. Toevaluate the effect of different COD/N ratios and the addition ofreal anaerobic effluent on the anammox process, the profiles ofnitrogenous compound concentrations were divided into three

phases: the control phase (Phase I), the operational phase, with dif-

ferent COD/N ratios due to glucose addition (Phase 2), and the realanaerobic effluent addition phase (Phase III).

3.1.1. Anammox cultivation phase (Phase I)

The first 323 days of operation corresponded to the anammoxenrichment and cultivation period after inoculation of biomass

(Phase I). During this phase, the reactor showed an average pHvalue of 7.3 0.8, a steady temperature of 35 C, and DO between0 and 0.5 mgL1. The ammonium and nitrite concentrations inthe influent ranged from, respectively, 100 mgL1 to 150 mgL1

and 150 mgL1 to 200 mgL1 due to variations in the preparationof culture medium (but the nitrite to ammonium ratio applied waskept around 1.31.4). Over the monitoring period in Phase I, theaverage removal efficiencies for ammonium and nitrite were 92%

and 97%, respectively (Fig. 1b and c), and the average nitrate pro-duction value was 39.6 mgL1 (Table 2).

Table 1

Characterization of the anaerobic effluent from a UASB reactor that treats municipal

wastewater.

Compound Unit Median value

pH 7.2 (0.1)a

Conductivity lscm1 744 (64)

DO mgL1 0.3 (0.1)

Temperature C 25.7 (2.3)

Total BOD mgL1 72 (22)

Total COD mgL1 150 (92)

Soluble COD mgL1 80 (35)

TSS mgL1 71 (43)

VSS mgL1 51 (30)

Total nitrogen mgL1 46 (8)

TNK mgL1 45 (8)NH4

+ mgL1 27 (4)

NO2 mgL1 0.0 (0.0)

NO3 mgL1 0.1 (0.1)

PO43 mgL1 6.9 (4.0)

SO42 mgL1 14.2 (4.5)

a The values in brackets are standard deviation.

C.D. Leal et al. / Bioresource Technology 211 (2016) 257266 259
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The stoichiometric coefficient (consumption of N-NO2

/consumption of N-NH4+) determined during Phase I was 1.46

(as shown in Table 2), which is close to that (1.32) reported for

an anammox reaction by Strous et al. (1998). Quan et al. (2008)reported a coefficient of 1.46 in an UASB reactor operated in anam-

mox conditions. The average value found for the coefficient of N-NO3 production/N-NH4

+ consumption was 0.34, slightly higher than

the one reported byStrous et al. (1998),of 0.26.Date et al. (2009)reported a coefficient of 0.33 (production of N-NO3

/consumptionof N-NH4

+) in an upflow reactor with biomass from a wastewater

treatment tank from a pig production facility. A similar result(0.36) was reported byPereira et al. (2014).

The results obtained in Phase I demonstrated that the anammoxprocess was established in the SBR, thus confirming that the two

(a)

(b)

(c)

Fig. 1. (a) Performance of the SBR during operation with autotrophic medium (Phase I, anammox cultivation), increasing influent COD/N ratios (Phase II, glucose addition),

and the addition of real anaerobic effluent (Phase III). Box plot of ammonium (b) and nitrite (c) removal efficiencies of the SBR during operation with autotrophic medium

(Phase I), increasing COD/N ratios (Phase II), and the addition of real anaerobic effluent (Phase III).



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inocula used and the operational conditions applied were suitable

for the enrichment and cultivation of anammox biomass. Thisphase was considered the control phase, and the nitrogenremoval values were used as references for comparison withresults obtained from subsequent phases.

3.1.2. Performance of the SBR during operation with different COD/N

ratios (Phase II)

For 131 days, from day 351 to 482 (Phase II), the reactor was

operated with increasing influent COD/N ratios due to glucoseaddition. To perform this experiment, the concentrations of ammo-nium and nitrite in the autotrophic medium were reduced to

60 mgL1 and 80 mgL1, respectively, (while the nitrite to ammo-nium ratio was kept around 1.31.4) for COD/N ratios of 0.7, 1.4,2.8, and 3.5.Fig. 1shows the concentrations of nitrogenous com-pounds in the influent and effluent of the SBR in Phase II. Increas-ing COD/N ratios were found to negatively affect ammonium

removal efficiency (Fig. 1b) and to partially inhibit the anammoxprocess (as confirmed by the mass balance results shown inTable 2). This was most notable for the COD/N ratio of 3.5 (CODof 490 mgL1), when the average ammonium removal efficiency

values dropped sharply to 21.7%. For this reason, the test wasstopped and the reactor was fed only with autotrophic medium(without glucose) for 33 days, from the 415th to the 448th day,

to recover nitrogen removal efficiency (this period was called therecovery period, or Phase R). The concentrations of nitrite and

ammonium in the autotrophic medium were reduced to 30 mgL

1

and 30 mgL1, respectively, to carry out the test with COD/N ratios

up to 5.0 after the recovery period (since COD was 300 mgL1 andnitrogen was 60 mgL1). Thus, in this period the nitrite to ammo-nium ratio applied was 1.0. During Phase II, the average pH valuewas 6.83 0.25, which was lower than the pH values observed in

Phase I (7.4 0.8); however, the pH values remained within theideal range for anammox bacteria, i.e., between 6.7 and 8.3(Strous et al., 1999). The nitrate concentrations in the SBR effluentwere also monitored during Phase II, and the average value was

15.8 mgL1.Regarding nitrite removal efficiency in Phase II, the results

showed that the average values remained close to 99%, indicatingthat nitrite removal was not affected by increasing COD/N ratios

applied to the reactor (as shown in Fig. 1c). The nonparametricKruskalWallis statistical test did not detect differences between

these values and those obtained during Phase I, followed by multi-ple comparisons of medians (a= 5%; p= 0.1531). However, forammonium removal efficiency, statistical differences betweenthe average values obtained from Phase II and Phase I weredetected by KruskalWallis test (a= 5%; p = 0.01356). In addition,for multiple comparisons of median values, the KruskalWallis test

(a= 5%) was performed and statistical differences were observedbetween periods with COD/N ratios of 0.7 and 1.4 (p= 0.0412),0.7 and 2.8 (p= 0.0453), 0.7 and 3.5 (p= 0.0167), 0.7 and the recov-ery phase (p= 0.0102) and 0.7 and 5.0 (p= 0.0201). Thus, the addi-

tion of glucose (at concentrations of 195 mgL1, 390 mgL1,487.5 mgL1 and 300 mgL1, corresponding to COD/N ratios of

1.4, 2.8, 3.5 and 5.0, respectively) affected ammonium removal effi-ciency and consequently the anammox process. Moreover, these

results indicate that the COD concentration of 487.5 mgL1 wasthe one that severely affected and inhibited the anammox processsince ammonium removal was significantly reduced (as shown inFig. 1b) and the color of the biomass changed from brownish-

yellow to a darker color (as shown in Supplementary Fig. S1).The nitrogen mass balance was calculated for all operational

phases to evaluate the participation of different processes (Table 2).The results indicated that the addition of glucose at different con-

centrations (during Phase II) gradually reduced the participation ofanammox process in the nitrite removal (from 87% at COD/N ratioof 0.723% at COD/N of 3.5) while favored the denitrification pro-cess, being the highest consumption of nitrite by denitrification

was observed at the COD/N ratio of 3.5 (Table 2). Nitrate removalduring Phase II was also observed, being the highest nitrate con-sumption via denitrification was observed at COD/N ratio of 2.8(Table 2).

The COD was monitored during Phase II to determine whetherthere was glucose consumption (COD removal) by other bacterialgroups or whether COD was accumulating inside the reactor. Theaverage COD removal efficiencies in each of the steps (shown in

Fig. 2) were 71% (COD of 97.50 mgL1, COD/N = 0.7), 82% (COD of195 mgL1, COD/N = 1.4), 62% (COD of 390.0 mgL1, COD/N = 2.8), 16% (COD of 487.50 mgL1, COD/N = 3.5), and 55% (CODof 300.00 mgL1, COD/N = 5.0), indicating that COD was being con-

sumed inside the reactor probably by denitrifying bacteria (con-firming the mass balance results).

Phase II may be viewed as the most critical phase of the wholestudy, as it was the one in which nitrogen removal performance by

Table 2

Nitrogen mass balance evaluation of participation of different processes.

Phase COD/N

ratio

NH4+-N removal

(mg N/L)a*SD NO2-N removal (mg N/L) NO3

-N (mg N/L) COD influent

(mg/L)

COD removal

(mg/L)

Stoichiometrye

Anammox Denitrification Productionb Removalc Finald

I 0 116.0 33.6 169.4 0.0 39.6 0.0 39.6 0.0 0.0 1:1.46:0.34

II 0.7:1 49.1 5.5 71.7 10.9 16.7 5.3 11.5 97.5 67.8

1.4:1 46.4 9.0 67.8 1.0 15.8 6.8 9.0 195.0 162.2

2.8:1 43.2 5.3 63.1 16.9 14.7 11.0 3.7 390.0 261.03.5:1 11.4 0.9 16.6 61.6 3.9 1.1 2.8 487.5 89.2R** 35.0 16.9 51.2 6.1 11.9 5.3 6.6 0.0 0.0

5.0:1 31.5 8.0 37.7 1.4 9.0 3.3f 12.3 300.0 174.4 1:1.30:0.31

III A 3.0:1 31.7 15.8 34.8 0.3 6.3 0.9 5.4 238.2 204.4 1:1.10:0.20

III B 3.5:1 48.5 10.1 82.4 32.1 9.7 0.9 8.8 172.2 133.1 1:1.70:0.20

* SD: Standard deviation.** Recovery period (without glucose).

a Mean values.b Values denote nitrate produced via anammox process.c Values mean nitrate removed by denitrification.d Values are the nitrate concentrations determined in the effluent.e The stoichiometry of removed NH4

+-N: NO2-N: produced NO3-N obtained during Phase I (control phase) was used to calculate the nitrite removal via anammox and

denitrification processes during Phase II (COD/N ratios of 0.7, 1.4, 2.8, 3.5; and R period). For other periods the stoichiometry used is shown in the table.f Negative value indicates that nitrate balance did not close, as more nitrate was determined in the effluent.

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the anammox process was significantly reduced in comparisonwith Phase I (Fig. 1b and Table 2). After testing the COD/N ratioof 3.5, the reactor underwent a recovery period with no organic

compound (glucose) added. During recovery phase and the subse-quent step (COD/N ratio of 5) as well, the anammox process wasreestablished in the reactor (as can be seen by the increase innitrite removal by this process, showed inTable 2).

3.1.3. Performance of the SBR during operation with real anaerobic

effluent (Phase III)

For 57 days, from days 483 to 540 (Phase III), the reactor was

operated with real anaerobic effluent. The effluent was taken froma UASB reactor that treats municipal wastewater and showed med-ian COD values of 150 mgL1 and ammonium concentration of

27 mgL1 (seeTable 1), corresponding to a COD/N ratio of 5.5.Over the Phase III monitoring period, the average pH inside the

reactor was 6.9 0.4, while the temperature was kept at 35 C andthe dissolved oxygen ranged from 0.0 to 0.4. The results of the SBRperformance during Phase III are presented in Fig. 1. Phases IIIA

and IIIB had different results for ammonium and nitrite removalefficiencies (Fig. 1b and c, respectively). In step A, in which thereactor was operated with real anaerobic effluent diluted withautotrophic medium (containing 50 mgL1 of NO2

-N and

30 mgL1 of NH4+-N), instability of the anammox process was

observed, as nitrite was completely consumed (the averageremoval efficiency was 99.8%, Fig. 1c), while the average ammo-

nium removal efficiency was 51% (Fig. 1b). These results mightindicate that there was not sufficient nitrite available for the anam-

mox reaction. In fact, in this step (IIIA) the nitrite to ammoniumratio applied was below 1.0 and therefore was not completely

favorable to the anammox. Moreover, some denitrificationoccurred in this phase (as shown in Table 2) which contributedto leave no terminal electron acceptor for anammox. For this rea-son, in step B, the reactor was operated with real anaerobic effluent

(undiluted) supplemented with 120 mgL1 of nitrite, so that theanammox bacteria would have sufficient electron acceptors avail-able to oxidize the ammonium.Tang et al. (2010)studied the inhi-bition of the anammox process when treating effluent with high

COD concentrations (from 50 to 700 mgL1), and observed thatanammox reactions were inhibited by denitrifying bacteriabecause they competed for electron acceptors. The authors added40 mgL1 of nitrite to the reactor to favor the anammox process.

In Phase IIIB, in which anaerobic effluent supplemented withnitrite was added to the reactor, the average ammonium removal

efficiencies were higher (85%) than in Phase IIIA (51%) ( Fig. 1b).Statistical differences were observed between these values whenthey were compared by MannWhitney test (a= 5%;

p= 0.00016). By the end of Phase IIIB, an increase in ammoniumremoval efficiencies to higher than 95% was observed.

Previous studies have shown that anammox bacteria enrichedin reactors fed with organic compounds such as propionate and

acetate are capable of competing with denitrifying heterotrophicbacteria for the electron acceptor (nitrite) because of their affinityfor the substrate (Kartal et al., 2012). Anammox bacteria have verylow growth rate and cellular yield (coefficient Y= 0.066 0.01)when compared to denitrifying heterotrophic bacteria

(Y= 0.27 0.3), according to Strous et al., 1998. Therefore, denitri-fying bacteria may grow at a higher rate when organic compounds

are combined with ammonium and nitrite in the wastewater beingtreated.

Nitrate production (via anammox process) was monitored dur-ing Phase III, and the average values obtained were 6.3 and9.7 mgL1 in steps A and B, respectively (Table 2). These valueswere lower than those obtained in Phases I and II. This might be

explained by an increase in denitrifying activity following the addi-tion of anaerobic effluent to the SBR. In fact, the mass balanceresults (Table 2) confirmed this observation and showed that 28%of the nitrite added to the reactor was consumed by denitrificationprocess (in Phase IIIB).

A COD consumption assessment was performed during PhaseIII, and the results varied depending on the composition of theanaerobic effluent used to feed the SBR. The average COD value

determined for the anaerobic effluent was 208.44 54 mg

L1

.During Phase III, COD removal efficiency was nearly 85% in stepA and 74% in step B (Fig. 2). Therefore, along with the anammoxprocess, heterotrophic denitrification was also taking place insidethe reactor (as shown in Table 2), as a large part of COD (from

the real anaerobic effluent) was consumed, and that COD removalexplained the high nitrite consumption, and, consequently, thehigher observed stoichiometric coefficient (N-NO2

/N-NH4+) of 1.7

(Table 2). In Phase IIIB, nitrite consumption via denitrificationwas 28% while via anammox was 72%, indicating that denitrifica-tion occurred in the reactor but anammox process prevailed.

The results from Phase III suggest that the anammox process

can be applied to the post-treatment of anaerobic effluents withhigh COD and low ammonium concentrations in order to removenitrogen. An important aspect of the present study was the appli-cation of the anammox process to the treatment of real anaerobic

Fig. 2. Box plot of COD removal efficiencies at different COD/N ratios during Phase II (glucose addition) and Phase III (addition of anaerobic effluent amended with nitrite).



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effluent with COD/N ratios between 4 and 6, while most other

studies have applied COD/N ratios of less than 3, except for Niet al. (2012), who also applied COD/N ratios of up to 4 (400 mgL1

COD). However, in their study, the authors observed inhibition ofthe anammox process with COD/N ratios of 3 and above. As faras we know, this is the first study that operated an SBR (with

anammox enriched biomass) using real anaerobic effluent (unfil-

tered and undiluted), rather than synthetic effluent or dilutedeffluent, to a reactor with pure anammox sludge (as was done inmost of the previous studies, such as Snchez-Guilln et al.,

2014; Jenni et al., 2014). This is another highlight of the presentstudy, since anammox bacteria were able to coexist with denitrify-ing bacteria as reported in the literature (Molinuevo et al., 2009;Oshiki et al., 2011; Kartal et al., 2012). Furthermore, the biomass

developed in the reactor was capable of simultaneously removingnitrogen and COD.

3.2. Bacterial community structure in the SBR before and after the

addition of glucose and real anaerobic effluent

DGGE profiles and band patterns from biomass developed in the

SBR and sampled at day 349 (after Phase I), day 482 (after Phase II,with glucose addition), day 448 (recovery phase-R, without glucose

addition), and day 558 (Phase III, after the addition of anaerobiceffluent) were evaluated (Fig. 3). The bacterial community struc-ture changed after the addition of glucose (Phase II) and anaerobic

effluent (Phase III) to the reactor (Fig. 3a).Representative bands were excised, and the DNA was

sequenced to identify microorganisms present in each phase ofreactors operation (Table 3). Most of the DGGE bands yielded

sequences related to those of bacteria within the phyla Proteobac-teria, Chloroflexi, andPlanctomycetes.

DGGE results showed that bands A and H, with sequences clo-sely related to the anammox bacteria Candidatus Brocadia carolin-iensis and Candidatus Brocadia sinica, respectively, were found in

every phase of the study (Fig. 3a andTable 3). These species arecommonly found in wastewater treatment systems (Hu et al.,2010). Ca. Brocadia sinica has been reported in the biomass of

anammox reactors from China, Japan, and Germany, indicatingthe worldwide distribution of these bacteria (Oshiki et al., 2011).

Physiologic characteristics of the species Ca. Brocadia sinicawere compared to the characteristics ofCa. Brocadia anammoxidansandCa. Kuenenia stuttgartiensisbyOshiki et al. (2011). They found

that Ca. Brocadia sinicahad a higher growth rate with less affinity

for the substrate compared to the other two species studied. In

their study, many experimental conditions were tested, such astemperature, dissolved oxygen, pH, salinity, and inhibition byorganic compounds (acetate, propionate, glucose, and methanol).Oshiki et al. (2011) concluded that in reactors under adverse

conditions and high ammonium and nitrite loads, Ca. Brocadiasinicaprevailed over other anammox bacteria. This was the casein the present study since band H (closely related to Ca . Brocadiasinica) were favored in the absence of glucose and prevailed in

the reactor after the addition of the anaerobic effluent.Bacteria, within the phylum Chloroflexi, were observed in the

present study (DGGE bands D, F, L, M, O, and P,Table 3). This phy-lum includes bacteria with diversified metabolism (Hug et al.,2013) and are frequently found in anammox reactors (Cho et al.,2010; Costa et al., 2014, andPereira et al., 2014). They can degradestarch, sugars and peptides (Hug et al., 2013), and thus they mightbe involved in COD removal observed in the present study.

Fig. 3. Bacterial community analysis by denaturating gradient gel electrophoresis (DGGE). (a) DGGE profile of the bacterial community in biomass sampled at day 349 (I)

(after Phase I, anammox cultivation), day 482 (II) (after Phase II, glucose addition), day 448 (R) (after 35 days without glucose-recovery phase), and day 558 (III) (Phase III,after the addition of anaerobic effluent). (b) Dendrogram based on the DGGE profiles.

Table 3

Identification of DNA band sequences obtained from DGGE.

Band RDP classifier Blast Similaritya

(%)

Acc. No.

A Ca.

Brocadiaceae

Ca. Brocadia

caroliniensis

98 KF810110.1

B, J Rhodocyclaceae Denitratisoma

oestradiolicum

97 KF810114.1

C Myxococcales Myxococcales 92 FJ552616.1D Chloroflexi Anaerolineaceae 97 HE648186.1

E Rhodospirillales Acetobacteraceae 91 EU193085.1

F Chlo ro flex i C al di li nea sp. 84 HQ043268.1

G Acetobacteraceae Acetobacteraceae 93 EU921207.1

H Ca. Brocadiasp. Ca. Brocadia sinica 99 AB565477.1

I Chlorobi Chlorobi 88 KJ941776.1

K Actinomycetales Propioniferaxsp. 90 KP419698.1

L Anaerolineaceae Chloroflexi 97 JF703582.1

M Caldilinea sp Chloroflexi 94 AY921707.1

N Planctomycetes Planctomycetales 80 FJ517124.1

O Thermoflexussp. Chloroflexi 95 AY921707.1

P Chlo ro flex i C hlor oflexi 96 AY921913.1

a Percentages indicate the similarity between the DGGE band sequences and the

closest matched sequences in GenBank. Words in bold indicate: DNA bands A and H

related to anammox bacteria (Brocadia); bands B, J and K are related to bacteria able

to degrade glucose and might be involved in COD removal.

http://-/?-http://-/?-


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Within the phylumProteobacteria, DGGE bands B and J (found in

every phase of this study), yielded sequences closely related to thatofDenitratisoma oestradiolicum, which is a denitrifying bacteria iso-lated from a municipal wastewater treatment plant in Germany(Fahrbach et al., 2006). This bacterium can degrade 17 beta-

estradiol as a carbon source, oxidizing it into CO 2 and H2O, whileNO3

is reduced to N2O and N2 (Fahrbach et al., 2006). Band K inthe DGGE profile showed sequence related to bacteria of the genus

Propioniferax (Actinomycetales). They are facultative anaerobicmicroorganisms that can ferment glucose and produce propionicacid and reduce nitrate as well (Yokota et al., 1994). Therefore,bands B, J, and K might be involved in the degradation of glucose

added to the reactor and consequently in COD removal.The changes in bacterial community structure, during different

phases of reactor operation, were investigated by using 16SrDNA-DGGE analyses. The similarities between the DGGE profiles foreach sample were calculated and visualized as a dendrogram

(Fig. 3b). Two distinct clusters (with 49.9% similarity) were identi-fied (Fig. 3b). The first cluster consists of samples I (biomass takenfrom the SBR after Phase I, day 349) and II (sampled at day 482after the increasing COD/N ratios) which had high similarity with

each other (above 85%). This result apparently indicates that the

addition of glucose did not change the bacterial community. How-ever, when the DGGE profile of sample R (biomass sampled at day

448, after 35 days without glucose) was compared with sample II

(biomass sampled at day 482 after glucose addition), changes inthe bacterial community structure were observed indicating thatthe addition of glucose was the driving force for bacterial selection.The presence of glucose selected against bands H, M, N, O, and P

which were related to anammox bacteria and Chloroflexi (seeFig. 3a andTable 3). These DNA bands were prominent in the sub-sequent phases, indicating that they were favored in the absence of

glucose and prevailed after the addition of the anaerobic effluent(Fig. 3a). Members of Chloroflexi can utilize microbial productsderived from cell decay (Okabe et al., 2005). This seemed to bethe case in the SBR after the stress caused by the addition of highconcentrations of COD.

The second cluster consists of samples R (biomass sampled atday 448, without glucose) and III (sampled at day 558 after theaddition of anaerobic effluent) which were more similar to eachother (81.1% similarity) than to the other profiles (samples I andII) (Fig. 3b). This might be explained by the fact that the anaerobic

effluent had low concentration of biodegradable organic com-pounds (median value for soluble COD was 80 mg L1) and thusthis condition was similar to the Phase R (recovery period without

glucose). Moreover, the addition of anaerobic effluent changed the

bacterial community profile and increased the diversity inside thereactor, since the Shannon diversity indices for samples I, II and III

(a)

(b)

Fig. 4. (a) Nitrogen removal rate and abundance of anammox, denitrifying (inferred from the nosZgene), and total bacterial populations in the biomass of the SBR in Phases I(anammox cultivation), II (after glucose addition), and III (after anaerobic effluent addition). (b) Relative abundance (in percentage) of anammox bacteria and denitrifiers in

relation to total bacterial populations.



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were 2.49, 2.50 and 2.82, respectively. The addition of anaerobi-

cally pre-treated municipal wastewater to the SBR did not selectagainst anammox bacteria, on the contrary, it allowed for the coex-istence of anammox, denitrifying bacteria and Chloroflexi.

The presence of denitrifying bacteria in the SBR was also con-firmed by qPCR (Fig. 4a and b). Higher concentrations of denitrify-

ing bacteria (as measured by nosZgene abundance) were observed

in the SBR biomass with increasing COD/N ratios (Phase II, withglucose addition) and after the addition of real anaerobic effluent(Phase III). These results indicate that the addition of glucose and

the anaerobic effluent favored the enrichment of denitrifiers andincreased their concentration threefold (from 2.57 109 to7.57 109 copies/g of sludge) (Fig. 4a). However, the concentrationof anammox bacteria (as measured by 16S rRNA gene abundance)

did not change with an increase in COD or application of anaerobiceffluent, indicating that long-term operation of the SBR enrichedand sustained the anammox population (ranging from 2.08 109

to 1.90 109 copies/g of sludge). Ni et al. (2010), investigating ananammox UASB reactor, observed an anammox concentration of4.6 108 copies/g of sludge when the reactor reach a nitrogenremoval efficiency of 94%.

The relative abundances of anammox bacteria and denitrifiersin relation to total bacteria are presented inFig. 4b. The results alsoshowed that the addition of glucose and the real anaerobic effluent(Phases II and III, respectively) changed the bacterial community

by increasing the abundance of denitrifiers more than fourfold inrelation to the anammox population.

4. Conclusions

High concentrations of COD (above 487 mgL1) inhibited theanammox process. However, a COD/N ratio of 5.0 (with COD con-centrations up to 300 mgL1) did not inhibit the process and

allowed for the coexistence of anammox and denitrifying bacteria.High COD, nitrite, and ammonium removal efficiencies (80%, 90%,and 95%, respectively) were obtained with the addition of realanaerobic effluent to the SBR. This changed the bacterial commu-

nity structure and selected for DNA sequences related to Brocadiasinica andChloroflexi. Thus, the feasibility of applying the anammoxprocess to nitrogen removal from anaerobically pre-treated munic-ipal wastewater was demonstrated.

Conflict of interest

All authors declare that they have no conflict of interest.

Acknowledgements

We are thankful to the following brazilian agencies: Coor-

denao de Aperfeioamento de Pessoal de Nvel Superior (CAPES),to Fundao de Amparo a Pesquisa do Estado de Minas Gerais(FAPEMIG) and to Conselho Nacional de Desenvolvimento Cient-fico e Tecnolgico do Brasil (CNPq).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, athttp://dx.doi.org/10.1016/j.biortech.2016.03.

107.

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