reseacharicle - hindawi

Research ArticleKeeping a Completely Autotrophic NitrogenRemoval over Nitrite System Effective in Treating LowAmmonium Wastewater by Adopting an Alternative Lowand High Ammonium Influent Regime

Qinglong Chang WeigangWang Jie Chen and Yayi Wang

State Key Laboratory of Pollution Control and Resources Reuse College of Environmental Science and EngineeringTongji University Siping Road Shanghai 200092 China

Correspondence should be addressed to Yayi Wang yayiwangtongjieducn

Received 19 January 2018 Accepted 6 March 2018 Published 17 April 2018

Academic Editor Shijian Ge

Copyright copy 2018 QinglongChang et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

An alternative low and high ammonium influent regime was proposed and adopted to keep a completely autotrophic nitrogenremoval over nitrite (CANON) effective when treating low ammonium wastewater Results show that by cyclic operating atan alternative low and high ammonium concentration for 10 days and 28 days the CANON system could effectively treat lowammonium wastewater Excessive proliferation of nitrite oxidizing bacteria (NOB) under low ammonium environment was stillthe challenge for the stable CANON operation but with 28 days of a high ammonium treatment combined with a sludge retentiontime control the NOB overproliferated in the low ammonium operational period could be under control Specifically when thenitrite oxidation rate reached 8 gNm3h theCANONsystem should enter the high ammonium influent operatingmode 16S rDNAhigh-throughput sequencing results show that the appropriate sludge discharging provided an environment favoring CandidatusJettenia

1 Introduction

Anaerobic ammonium oxidation (anammox) is a new path-way of autotrophic biological nitrogen removal for wastew-ater treatment with the advantages of less aeration con-sumption low biomass production and carbon savings[1 2] Complete autotrophic nitrogen removal over nitrite(CANON) system which combines anammox with partialnitrification in a single reactor is one of the most promisingmethods to achieve energy neutral or even positive inwastewater treatment plants (WWTPs) [3 4] In this processammonium is first partially oxidized to nitrite by aerobicammonium oxidizing bacteria (AOB) through controllingdissolved oxygen (DO) at a low concentration The residualammonium is oxidized to nitrogen gas with the generatednitrite by anammox bacteria subsequently [5 6] Comparedwith the traditional nitrification and denitrification systemsCANON process can be a more promising and economic

technology for wastewater treatment because of 63 less oxy-gen consumption no requirement of biodegradable organiccarbon addition and less N

2O emissions [5 6]

So far CANON systems have been mostly applied totreat high ammonium wastewaters such as landfill leachateswine wastewater and reject water in WWTPs (known assidestream anammox) [7ndash12] This is because high ammonia(as well as high free ammonia (FA)) high temperature anda low DO control favor selecting both AOB and anammoxbacteria as the dominant functional microorganisms thusmaintaining the stable partial nitrification and anammoxprocesses Specifically the physiological differences betweenAOB and nitrite oxidizing bacteria (NOB) should be consid-ered that is environmental factors such as pH temperatureFA free nitrous acid (FNA) and DO must be controlledfavoring AOB proliferation in the partial nitritation step toproduce an anammox-suited substrate [13]

HindawiBioMed Research InternationalVolume 2018 Article ID 9536761 9 pageshttpsdoiorg10115520189536761

2 BioMed Research International

When treating mainstream wastewater that is character-izedwith low ammonium and ambient temperature stressingNOB and maintaining the activity of anammox bacteria arethe two key factors determining the CANON performance[14] Indeed substantial efforts have been made to optimizethe treatment process for a steady performance in CANONsystems when treatingmainstreamwastewater Han et al [15]used screens to separate flocs and granules in mainstreamdeammonification to wash out NOB and they found thatover 80 of NOB was washed out and up to 70 of nitrogenremoval efficiencies was achieved Similarly Malovanyy etal [14] tried integrated fixed film activated sludge reactorfor nitrogen removals from municipal wastewater using adeammonification process by intermittent aeration with ahighDO set-point (15mgL) and decreasing nonaerated time(30min) NOB was successfully out-selected However othereffective and operative approaches to ensure the effectivenitrogen removal performance of CANON systems are stillurgently required especially when treating low ammoniumwastewater

In this study a novel operational pattern for CANONsystem treating low ammonium wastewater was developedby alternatively inflowing low ammonium concentration(mimic mainstream wastewater) and high ammonium con-centration (mimic sidestream wastewater) The influentexchange frequency and the key controlling factors foreffective CANON operation were examined To elucidate themicrobial structure of functional bacteria and their influenceto the nitrogen removal performance the composition of thebacterial communities in the CANON system during oper-ational period was analyzed by 16S rDNA high-throughputsequencing technology The proposed operational strategyhopes to help CANON processes to be effectively applied inmunicipal wastewater treatment

2 Materials and Methods

21 Reactor Configuration A lab-scale sequencing batchreactor (SBR) with a working volume of 25 L was used in thisstudy The SBR was set in a thermostatic water bath to keeptemperature at 30∘CThe experimental biomass in the reactorwas mixed by a propeller stirrer at 100 rpm An aerationdevice linked with an air pump was fixed at the bottom of theSBR to provide oxygen for partial nitrification Air wasregularly supplied to the SBR by an air pump

22 Operational Strategies An alternative low and highammonium influent regime was adopted in the experimentAs shown in Figure 1 the whole experimental period wasdivided into four operational phases according to the influentammonium concentration Phase high I (0ndash60 d) Phase lowI (61ndash148 d) Phase high II (149ndash256 d) and Phase low II(257ndash285 d) Phases high I and II were operated at a highammonium concentration (approximately 240mgNH

4

+-NL) while Phases low I and II (approximately 61mgNH

4

+-NL) were operated at low ammonium concentration Atypical SBR cycle lasts for 4 h or 8 h respectively at low orhigh influent ammonium concentrations (Table 1) By oper-ating in an alternative low and high ammonium influent

regime the NOB inhibition and the dominance of AOB andanammox bacteria in the CANON system can be ensured

23 Seed Sludge and Feeding Medium The seeding granularsludge (25 L) for the experimental CANON systemwas fromanother CANON system treating high ammonium salinewastewater in our laboratory (Figure S1(A)) [3]

Synthetic wastewater was continuously introduced intothe reactor using a peristaltic pump in the inflow periodand the pump was controlled by a liquid level controller tocontrol the water volume of 25 L The composition of thesynthetic wastewater was KH

2PO4005 gL CaCl

203 gL

MgSO4sdot7H2O 03 gL NaHCO

3125 gL FeSO

4sdot7H2O

000625 gL Na2EDTA 000625 gL and 125mLL of trace

elements solution The composition of the trace elements so-lution was prepared according to Lotti et al [16] NH

4Cl was

added to the feeding medium to reach a desired ammoniumconcentration in the influent The pH was maintained at 80at the beginning of each cycle by adding hydrochloric acidsolution (02M) or sodium hydroxide solution (02M)

24 Analytical Methods Water samples were collected andstored in a 4∘C refrigerator until analysis The concentrationsof NH

4

+-N NO2

minus-N and NO3

minus-N were measured regularlyaccording to the standard methods [17] The DO concen-tration and pH were detected using online probes (WTWMulti350i Germany)

25 Sample Collection DNA Extraction and PCR Ampli-fication Sludge samples were collected from the CANONsystem on days 60 79 124 125 148 257 and 285 (the daysbefore the change in the operating condition) and stored atminus20∘Cafter centrifugation Total genomicDNAwas extractedin triplicate from each sample using the Power Soil DNAIsolationKit (Sangon China) according to themanufacturerrsquosinstructions The quality of the obtained genomic DNAwas examined by 1 (wv) agarose gel electrophoresis andconcentration measured with NanoDrop spectrophotometer2000 (Thermo Scientific USA)

Polymerase chain reaction amplification of the V3-V4region of the 16S rDNA gene was then conducted usingprimers 341F (51015840-CCTACACGACGCTCTTCCGATCTN-31015840)and 805R (51015840-GACTACHVGGGTATCTAATCC-31015840) with thereverse primer containing 6-bp barcodes to tag each sam-ple (Majorbio Bio-Pharm Technology Co Ltd ShanghaiChina) PCR amplification and 16S rDNA high-throughputsequencing were performed according to our previous study[3]

3 Results and Discussion

31 The Stabilization of the CANON System at High InfluentAmmonium Concentration The CANON system operatedfor 285 days and the long-term nitrogen removal per-formance was shown in Figure 1 The biomass was ini-tially cultivated with high ammonium concentration (196plusmn 18mgL) to set up the CANON system (Figure 1 Phasehigh I) After acclimation for 32 days the TN removal rate

BioMed Research International 3

Table 1 Operating parameters of the CANON system

Phase High I Low I High II Low II

Periods (d) 0ndash60 61ndash148 149ndash256 257ndash285

Cycle time (h) 8 4 8 4

Influent FA (mgNL) 5ndash7 lt3 7ndash10 lt3

Influent NH4

+-N (mgL) 196 plusmn 18 77 plusmn 45 240 plusmn 21 61 plusmn 56Volumetric exchangeratio 05

DO (mgO2L) 02ndash04 02ndash04 02ndash04 04ndash06

Temperature (∘C) 30 30 30 30

Aeration mode

3 aeration and anoxic stagesin one SBR cycle(35min105minrespectively)

50-min aeration stage inone SBR cycle



0 50 100 150 200 250 3000

50

100

150

200

250

300

28525614860

Phaselow IIPhase high IIPhase low I

Nitr

ogen

conc

entr

atio

n (m

gL)

Time (d)

Phase high I

00

01

02

03

04

05

TNRR

(kg

Nm

3 d)

TN

RE (

)

100

80

60

40

20

0

NH4+ inf

NO2minuseff

TNRR

NH4+eff

NO3minuseff

TNRE

Figure 1 Profiles of nitrogen compounds (expressed as influent ammonium concentration effluent ammonium concentration effluent nitriteconcentration and effluent nitrate concentration) total nitrogen removal rate (TNRR) and total nitrogen removal efficiency (TNRE) of theCANON system over 285 days of operation

(TNRR) and efficiency (TNRE) reached 151 gNm3h and80 respectively

Thereafter (days 32ndash60) the TNRE decreased to 70 asthe influent NH

4

+-N concentration increased but the TNRRremained stable at approximately 026 kgNm3d indicat-ing that the studied CANON system had achieved steady-state nitrogen removal performance (Figure 1) Also theammonium oxidation rate (AOR) and the TN removal rateduring the aeration stage (NRR) reached 212 gNm3h and026 kgNm3day respectively and nitrite oxidation rate

(NOR) was below 3 gNm3h suggesting that the NOBproliferation had been controlled

32 The CANON Performance Operated at Alternative Lowand High AmmoniumConcentrations After a stable nitrogenremoval performance was achieved in Phase high I theCANON system adopted an alternative low and high ammo-nium concentration inflowing mode that is Phase low I andPhase high II (Figure 1)


0 10 20 30 40 50 60

0

5

10

15

20

25

Time (d)

In si

tu ac

tivity

(gN

m 3 d

)

00

01

02

03

04

05

06

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(a)

63 70 77 100 110 120 130 1400

5

10

15

20

25

30

Time (d)

02

04

06

08

10

In si

tu ac

tivity

(gN

m 3 h

)

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(b)

In si

tu ac

tivity

(gN

m 3 h

)

150 165 180 195 210 225 240 2550

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10Δ

NO3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(c)

In si

tu ac

tivity

(gN

m 3 d

)

260 265 270 275 280 2850

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(d)

Figure 2 Changes in AOR NOR NRR and ΔNO3

minusΔNH4

+ ratio of the CANON system over the whole operational duration ((a) Phasehigh I (b) Phase low I (c) Phase high II (d) Phase low II)

321 Operating at a Low Ammonium Concentrationin Phase Low I

(1) Variations in Nitrogen Removal Performance After theinfluent ammonium decreased the nitrogen removal per-formance of the CANON system was stable firstly (days61ndash74) and decreased thereafter (days 77ndash148) (Figure 1)Specifically when the influent NH

4

+-N was decreased to 77plusmn 45mgL on day 61 the TNRR immediately decreased from026 kgNm3d (day 60) to 017 kgNm3d (day 61) with adecrease percentage of 32 Luckily the AOR was still stableat 22 gNm3h for about 10 days at low influent ammoniumconcentration even being slightly higher than 212 gNm3hin Phase high I (Figures 2(a) and 2(b)) In contrast NRRdropped to 11 gNm3h being lower than 151 gNm3h ofPhase high I indicating that the low influent NH

4

+-N

concentration had a greater impact on anammox reactionthan on ammonia oxidation reaction

Notably NOR increased gradually to approximately 49gNm3h during days 61ndash74 at a low ammonium influent(Figure 2(b)) suggesting that anammox bacteria could notcompete with NOB for nitrite under the low influent NH

4

+-N concentration operation [6] As the inhibitory threshold ofFA for AOB andNOB is 8ndash120mgNL and 008ndash082 respec-tively [18] NOB are generally more sensitive to FA than AOBand can be outcompeted by AOB under a high ammoniumenvironment (generally high FA as well) However as shownin Table 1 at low NH

4

+-N concentrations the competitivecapacity of AOB for oxygen was notmuch greater than that ofNOB due to the low FA (below 3mgNL) in Phase low I inthis study Nevertheless during days 61ndash74 in Phase low Ithe TNRE of the CANON was stable at 75 as a whole Alsowhen comparedwith Phase high IΔNO

3

minusΔNH4

+ increased


but was stable at approximately 023 (Figure 2(b)) indicatingthat theNOB abundance or activities were stable during these14 days

During days 74ndash124 (Phase low I) the nitrogen removalperformance of the CANON continuously decreased (Fig-ure 1) For instance the TNRR and TNRE decreased to005 kgNm3d and 23 respectively Although theAORwasstill stable at 18 gNm3h the NOR increased sharply to 146gNm3h with the ΔNO

3

minusΔNH4

+ being high at 069 (Fig-ure 2(b)) Also the nitrate concentration in the effluentincreased to 274mgL (Figure 1) This is possibly becauseanammox bacteria was not able to compete with NOBfor nitrite at a low influent NH

4

+-N concentration whichuntimely exposed anammox bacteria to a famine scenario

It should be noted that the CANON reactor was shutdown from days 78 to 101 because of the time control-ling breakdown Then the NOR increased sharply from557 gNm3h on 102 d to 146 gNm3h on 124 d (Fig-ure 2(b)) Considering that NOB and AOB are prone tocolony in flocs and anammox bacteria tends to be aggregatedas granules we meshed the activated sludge of the CANONsystem to quickly recover the CANON performance Specif-ically 1 L mixed liquid was drawn from the CANON reactorand the flocs were removed using a screen with 80 meshTheleft granules were poured into theCANONreactor againwith1 L shortcut nitrifying sludge (mainly containing AOB) froma shortcut nitration reactor in our laboratory

After 7 daysrsquo recovery (day 130) the TNRR and TNREincreased to 017 kgNm3d and 645 respectively (Fig-ure 1) The AOR remained constant because of the low DO(02ndash04mgL) but NOR decreased by 60 from 146 gNm3h (day 124 in Phase low I) to 6 gNm3h (day 130)The NRR increased to 96 gNm3h and ΔNO

3

minusΔNH4

+

decreased to 024 with decreasing NOR (Figure 2(b)) Thisresult suggests that it was suitable to washout NOB andrecover the nitrogen removal performance in a short timethrough removing the flocs as AOB and NOB are mainlycolonized in flocs [15]

After day 130 in Phase low I the CANON reactordeteriorated again (Figure 1) Notably the NOR increased to8 gNm3h on day 140 and to 118 gNm3h on day 148(Figure 2(b)) Meanwhile TNRE decreased to 368 andΔNO3

minusΔNH4

+ increased to 05 (Figure 2(b)) indicatingthat NOB had proliferated again and competed nitrite withanammox bacteria It seems that controlling only DO at alow level could not sustain a steady shortcut nitrification[19]

(2)TheNORVariation Characteristics Remarkably there wasa linear relationship between NOR and operational days thatNOR increased linearly with the operational days (Figure 3)When NOR was below 8 gNm3h the NOR was graduallyincreased along with the operational days with a slope of039 However after NOR was beyond 8 gNm3h the NORsharply increased with a slope of 098 until the CANONSBR completely collapsed It seems that the NOR should becontrolled under 8 gNm3h to ensure the stable nitrogenremoval performance of the studied CANON system Thus

100 105 115 120 125 1304

6

8

10

12

14

16

Time (d)

y = 043x minus 379 (R2 = 098)

y = 001x minus 347 (R2 = 039)

NO

R (g

Nm

3d

)

Figure 3 Linear fittings of NOR and time of the CANON reactorduring the operation of days 103ndash108 and days 112ndash124 of Phase lowI

NOR was selected as an indicting parameter for CANONoperated at a low ammonium concentration

322 Operating at a High Ammonium Concentration in PhaseHigh II On day 149 the influent ammonium concentrationwas increased to 240 plusmn 21mgL again to recover the CANONperformance (Figure 1 Phase high II) The pH in the inflowwas still controlled at 80 and the influent FA concentrationwas about 7ndash10mgNL By improving the FA concentrationwe expected to stress the NOB proliferation and recoverthe functional bacteria such as AOB and anammox bacteriain the studied CANON Corresponding to the increasedinfluent ammonium the aeration time increased by 80 from300min to 540min for one day with the unchanged DOconcentration at 02ndash04mgL Then both the FA and DOconcentrations set in Phase high II were favorable to inhibi-tion of NOB [18 20 21]

(1) The Stable Nitrogen Removal Performance at High Ammo-nium Concentration During day 149 to day 158 whenoperating at a high ammonium concentration of approxi-mately 240mgL the nitrogen removal performance of theCANON system continued to increase with the TNRR andNRR up to 025 kgNm3d and 161 gNm3h respectivelyon day 158 (Figures 1 and 2(c)) Correspondingly theNOR and ΔNO

3

minusΔNH4

+ decreased to 67 gNm3h (below8 gNm3h) and 028 respectively (Figure 2(c)) indicatingthat NOB had been effectively inhibited at the high ammo-nium concentration and then anammox bacteria couldcompete with NOB for nitrite

However the nitrogen removal performance of theCANON system decreased from day 159 (Figure 1) Specif-ically the NRR decreased to 10 gNm3h on day 170 andtheNOR andΔNO

3

minusΔNH4

+ increased to 107 gNm3h and045 respectively (Figure 2(c)) These results illustrate thatNOBmight have been adapted to the high FA andproliferatedagain even at the high ammonia environment [22] Ourresults are somewhat in contrast to those of Wang and Gao


[23] who recovered the CANON system in 56 days fromthe excessive multiplication of NOB using simultaneous highammonium and nitrite concentration in the inflow Thedifferent results observed in this study with other studies arepossible due to the fact that it was difficult to keep the highnitrite in the studied CANON system as nitrite producedby AOB could be simultaneously or promptly be assumed byanammox bacteria in the CANON operational mode

(2) SRT Adjustment to Washout NOB Once the over prolif-erated NOB occurred in the CANON system it is difficultto inhibit NOB due to the low decay rate of NOB [24] Thisproblem can be resolved by discharging the NOB sludge [8]For example in Strass WWTP (Austria) separation of AOBNOB and anammox bacteria was handled by a hydrocycloneand washing out NOB was effectively achieved by controllingof the selected SRT of AOB and anammox bacteria [25 26]Mimicking this case from day 175 sludge discharging wasadopted to the CANON system to control the SRT of approxi-mately 60 dThen during days 175ndash202 the TNRR andTNREincreased to 029 kgNm3d and 635 (day 202) (Figure 1)respectively the NOR decreased to 26 gNm3h with theeffluent nitrate concentration decreased from 642mgL (day175) to 456mgL (day 202) (Figures 1 and 2(c)) Also theΔNO3

minusΔNH4

+ decreased to 02 on day 202 and NRRincreased to 23 gNm3h (Figure 2(c))

Although a part of NOB was washed out by the sludgedischarging the AOR also decreased due to the loss ofthe activated sludge Consequently the TNRR decreasedgradually with further discharge of sludge Specifically theAOR andNRR decreased to 134 gNm3h and 178 gNm3hrespectively on day 218 (Figure 2(c)) As shown in Figure S2the MLVSS of the studied reactor decreased gradually due toover discharge of the sludge As a result the amount andactivity of AOB and anammox bacteria decreased Howeverbecause NOB was less abundant in the biomass than AOBand anammox bacteria sludge discharging would lead to alower percentage of NOB in the residual CANON systemTherefore the NORwas still stable at 26 gNm3h (far below8 gNm3h) andΔNO

3

minusΔNH4

+ was also stable at 02 duringdays 202ndash218 (Figure 2(c)) To prevent the continued decreasein the TNRR the sludge discharging was stopped on day 219(Figure 1) With increasing MLVSS during days 219ndash256 theTNRR and TNRE were increased to 023 kgNm3d and 55on day 256 (Figure 1) and the NOR and ΔNO

3

minusΔNH4

+

could be stable at 33 gNm3h and 02 (Figure 2(c)) respec-tively

Taken together our results show that high ammoniumconcentration (240mgL) and controlled SRT (60 day)could improve the performance of CANON system throughdecreasing NOB to a low abundance in 28 days (days175ndash202)

323 Stability of the CANON System in a Low AmmoniumConcentration in Phase Low II After day 257 (in Phase lowII) the ammonium concentration was decreased to 61 plusmn56mgL again (Figure 1) to examine the stability of CANONsystem at low ammonium concentrations During day 257

to day 266 (approximately 10 days) the performance of theCANON system was stable TNRR and TNRE were stableat 015 kgNm3d and 70 (Figure 1) respectively Howeverafter day 267 the CANON system deteriorated again that isthe TNRR was lower than 01 kgNm3d and NOR increasedto 8 gNm3h after day 279 (Figures 1 and 2(d)) The opera-tional results during Phase low II confirmed that the systemwould deteriorate once NOR reached 8 gNm3h

In summary in Phase low I (day 61ndash74) and Phase low II(day 257ndash266) the CANON system could be stably operatedfor approximately 10 days at low ammonium concentrations(60mgL) with a relatively high TNRE (70) and loweffluent N concentrations (5mgNH

4

+-NL and 20mgTNL)(Figure 1)

324 Microbial Composition and Structure Variations withCyclic Low and High Influent Ammonium

(1) Bacterial Community Composition The composition ofthe bacterial communities in the CANON system was ana-lyzed by 16S rDNA high-throughput sequencing At phylumlevel the CANON system was dominated by ChloroflexiProteobacteria Planctomycetes and Chlorobi (Figure 4(a))Anammox bacteria and AOB the functional bacteria inthe CANON system were affiliated to Planctomycetes andProteobacteria respectively Chloroflexi and Chlorobi werealso extensively detected in other anammox systems [27]and Chloroflexi could provide structure support for sludgegranulation using the decayed anammox biomass [28 29]

TheN-related bacteriawereNitrosomonas-affiliatedAOBNitrospira-affiliated NOB and ldquoCandidatus Jetteniardquo anam-mox bacteria (Figure 4(b)) Chu et al [30] also found thatldquoCandidatus Jetteniardquo and Nitrosomonas were the dominantfunctional bacteria in their CANON system treating highammonium wastewater (500mgNLminus1) with the relativeabundances 168 and 201 respectively It should be notedthat there also existed Denitratisoma-affiliated denitrifyingbacteria in the CANON system Denitratisoma was reportedto be able to use 17b-oestradiol as the sole carbon source andenergy and electron donor to reduce nitrite to nitrous oxide[31]

(2) The Variations of N-Transformation Microorganisms Inorder to elucidate the variations in the abundances of the N-related bacterium Candidatus Jettenia Candidatus Kuene-niaNitrosomonas andNitrospirawere plotted in Figure 4(c)When theCANONsystemwas stable in Phase high I (day 60)Candidatus Jettenia andCandidatusKueneniawere the domi-nant anammoxbacteriawith the relative abundances of 356and 69 respectively However when the ammonium con-centration decreased in Phase low I Candidatus Jettenia out-competed CandidatusKuenenia and became the main anam-mox genera (day 79) Specifically from day 124 to day 148in Phase low I the relative abundance of Candidatus Jetteniaincreased from 591 to 144 This is primarily because theincreased AOB amount and decreased NOB amount aftersludge changing caused the increased nitrite concentration


ChloroflexiProteobacteriaPlanctomycetesChlorobiAcidobacteriaBacteroidetesActinobacteriaFirmicutesNitrospiraeArmatimonadetes

Bacteria_unclassifiedVerrucomicrobiaDeinococcus-ThermusSHA-109DeferribacteresCandidate_division_WS6GemmatimonadetesMicrogenomatesOthers

100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(a)

Anaerolineaceae_unculturedCandidatus_JetteniaSJA-28_norankNitrosomonasBlastocatellaPHOS-HE51_norankOM1_clade_norankDenitratisomaNitrospiraChloroflexi_unculturedBacteria_unclassifiedTK10_norankABS-19_norankEscherichia-ShigellaLimnobacterCaldilineaceae_unculturedAquicellaArmatimonadetes_norankLD29_norankCandidatus_KueneniaPseudomonasSHA-109_norank

ThermusTuricibacterStenotrophomonasCaldithrixOPB56_norankVariovoraxHaliangiumCandidate_division_WS6_norankPHOS-HE36_norankSyntrophaceae_unculturedHydrotaleaIgnavibacteriumTerrimonasGemmatimonadaceae_unculturedTepidamorphusDeltaproteobacteria_unclassifiedMicrogenomates_norankSaprospiraceae_unculturedChitinophagaceae_unculturedDB1-14_norankBrocadiaceae_unclassifiedOthers

100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(b)

0

10

20

30

40

50

60

C285C257C148C125C124C79

Relat

ive a

buda

nce (

)

Candidatus JetteniaCandidatus Kuenenia

NitrosomonasNitrospira

C60Time (d)

(c)

Figure 4 The microbial community taxonomic compositions in the studied CANON system (a) at the phylum level (b) at the genus level(c) changes in the relative abundance of Candidatus Jettenia Candidatus Kuenenia Nitrosomonas and Nitrospira


After being cultivated during Phase high II CandidatusJettenia was still the dominant anammox bacteria (days257ndash285) while the relative abundance ofCandidatusKuene-nia was at an extremely low level (025 and 002 on days257 and 285 respectively) For example the relative abun-dance of Candidatus Jettenia increased from 144 (day 148Phase low I) to 4532 (day 257 Phase high II) Obviously ahigh ammonium concentration had favored to enrich anam-mox bacteria As each genus of anammox bacteria has itsown special ecological niche [32] the higher relative abun-dance of Candidatus Jettenia than that of Candidatus Kue-nenia indicates that the present experimental condition wasmore suitable for Candidatus Jettenia

As shown in Figure 4(c) on days 124 (Phase low I) 148(Phase low I) and 285 (Phase low II) the relative abun-dance of Nitrospira (NOB) increased slightly because of thelow ammonium concentration However after the sludgechanging on day 124 the relative abundance of Nitrospiradecreased from 401 (day 124) to 057 (day 125) andNitro-somonas increased from 503 to 1332 The relative abun-dance of Nitrospira also decreased from 355 (day 148) to043 (day 257) after being cultivated at a high ammoniumconcentration in Phase high II suggesting that it was suitableto enrich AOB and inhibit NOB using sludge changing and ahigh ammonium concentration

Compared with Phases high I and low I Phases high IIand low II seem to be more robust in the nitrogen removalperformance (Figure 1) possibly because the relative abun-dance of anammox bacteria was high in Phases high IIand low II (Figures 4(b) and 4(c)) Our results suggest thatreplenishing anammox bacteria biomass into the CANONsystem could be an alternative strategy for stabilization ofanammox treatment performance

33 The Recommended Operation Strategy for Practical Oper-ation of CANON Systems According to our experimentalresults the alternative low and high ammonium influentregime was feasible for CANON system to treat a part of lowammonium wastewater It is recommended alternatively tooperate CANON system at low ammonium concentration for10 days and at high ammonium concentration for 28 daysAlso the NOR and sludge age (SRT) as two importantparameters were recommended to be below 8 gNm3h andapproximately 60 d in the present CANON systems

The proposed strategy can be realized if WWTPs havesludge digestion unit from which the higher ammoniuminfluent can be supplied Also several parallel CANON SBRunits are required so that when a series CANON SBRs treatmainstream wastewater other series can treat sidestreamwastewater (ie sludge digestion supernatant) for enhance-ment of AOB and anammox bacteria and inhibition ofNOB By this way CANON system can treat nitrogen con-taining wastewater continuously But to use this operationalregime successfully in mainstream CANON system thedifference between actual ammonium concentration in realWWTPs and our experiment must be considered Furtherresearch should be focused on improving the proportion ofthe low ammonium concentration treatment duration andovercoming low temperature in real municipal wastewater

Our strategy hopes to help opening a new possibility forCANON processes used in municipal wastewater (main-stream wastewater) treatment

4 Conclusions

An alternative low and high ammonium influent regimewas proposed and investigated to keep CANON stable whentreating low ammonium wastewater Alternatively operatingat a low ammonium concentration for 10 days and at ahigh ammonium concentration for 28 days was feasible forCANON to treat low ammonium wastewater NOR andsludge age as two important parameters should be con-trolled to maintain a stable operation NOR should be keptunder 8 gNm3h to prevent CANON deterioration To useCANON in mainstream successfully further studies areneeded to shorten the duration of operating at high ammo-nium concentrations and overcome low temperature

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (51522809 and 51378370)

Supplementary Materials

Figure S1 the anammox granule of seeding CANON sludge(A) seeding CANON sludge (B) anammox granule aftersieving on day 124 (C) flocs after sieving on day 124 (D)Figure S2 changes in the MLSS MLVSS and ΔNO

3

minusΔNH4

+

ratio over the operation of Phase high II Figure S3 rar-efaction curve Table S1 microbial community richness anddiversity index of sludge samples (Supplementary Materials)

References

[1] B Kartal N M De Almeida W J Maalcke H J M Op denCamp M S M Jetten and J T Keltjens ldquoHow to make a livingfrom anaerobic ammonium oxidationrdquo FEMS MicrobiologyReviews vol 37 no 3 pp 428ndash461 2013

[2] M Strous J A Fuerst E H M Kramer et al ldquoMissinglithotroph identified as new planctomyceterdquo Nature vol 400no 6743 pp 446ndash449 1999

[3] Y Wang J Chen S Zhou et al ldquo16S rRNA gene high-throughput sequencing reveals shift in nitrogen conversionrelated microorganisms in a CANON system in response to saltstressrdquo Chemical Engineering Journal vol 317 pp 512ndash521 2017

[4] X Zhang D Li Y Liang Y He Y Zhang and J ZhangldquoAutotrophic nitrogen removal from domestic sewage in MBR-CANON system and the biodiversity of functional microbesrdquoBioresource Technology vol 150 pp 113ndash120 2013

[5] A O Sliekers N Derwort J L Campos GomezM Strous J GKuenen and M S M Jetten ldquoCompletely autotrophic nitrogenremoval over nitrite in one single reactorrdquoWater Research vol36 no 10 pp 2475ndash2482 2002


[6] K A Third A O Sliekers J G Kuenen and M S MJetten ldquoThe CANON system (completely autotrophic nitrogen-removal over nitrite) under ammonium limitation interactionand competition between three groups of bacteriardquo Systematicand Applied Microbiology vol 24 no 4 pp 588ndash596 2001

[7] M Azari UWalter V Rekers J-D Gu andM Denecke ldquoMorethan a decade of experience of landfill leachate treatment with afull-scale anammox plant combining activated sludge andactivated carbon biofilmrdquo Chemosphere vol 174 pp 117ndash1262017

[8] A Joss D Salzgeber J Eugster et al ldquoFull-scale nitrogenremoval from digester liquid with partial nitritation and anam-mox in one SBRrdquo Environmental Science amp Technology vol 43no 14 pp 5301ndash5306 2009

[9] S Lackner E M Gilbert S E Vlaeminck A Joss H Hornand M C M van Loosdrecht ldquoFull-scale partial nitritationanammox experiencesmdashan application surveyrdquoWater Researchvol 55 pp 292ndash303 2014

[10] WR L van der StarW R AbmaD Blommers et al ldquoStartup ofreactors for anoxic ammoniumoxidation Experiences from thefirst full-scale anammox reactor in RotterdamrdquoWater Researchvol 41 no 18 pp 4149ndash4163 2007

[11] T Yamamoto K Takaki T Koyama and K Furukawa ldquoLong-term stability of partial nitritation of swine wastewater digesterliquor and its subsequent treatment by Anammoxrdquo BioresourceTechnology vol 99 no 14 pp 6419ndash6425 2008

[12] F Zhang Y Peng LMiao ZWang SWang and B Li ldquoA novelsimultaneous partial nitrificationAnammox and denitrification(SNAD) with intermittent aeration for cost-effective nitrogenremoval from mature landfill leachaterdquo Chemical EngineeringJournal vol 313 pp 619ndash628 2017

[13] S W H Van Hulle H J P Vandeweyer B D MeesschaertP A Vanrolleghem P Dejans and A Dumoulin ldquoEngineer-ing aspects and practical application of autotrophic nitrogenremoval from nitrogen rich streamsrdquo Chemical EngineeringJournal vol 162 no 1 pp 1ndash20 2010

[14] A Malovanyy J Trela and E Plaza ldquoMainstream wastewatertreatment in integrated fixed film activated sludge (IFAS)reactor by partial nitritationanammox processrdquo BioresourceTechnology vol 198 pp 478ndash487 2015

[15] M Han S E Vlaeminck A Al-Omari et al ldquoUncoupling thesolids retention times of flocs and granules in mainstreamdeammonification A screen as effective out-selection tool fornitrite oxidizing bacteriardquo Bioresource Technology vol 221 pp195ndash204 2016

[16] T Lotti R Kleerebezem Z Hu B Kartal M S M Jetten andM C M van Loosdrecht ldquoSimultaneous partial nitritationand anammox at low temperature with granular sludgerdquoWaterResearch vol 66 pp 111ndash121 2014

[17] APHA Standard Methods for the Examination of Water andWastewater American Public Health Association WashingtonDC USA 21st edition 2005

[18] A C Anthonisen R C Loehr T B S Prakasam and E GSrinath ldquoInhibition of nitrification by ammonia and nitrousacidrdquo Journal of the Water Pollution Control Federation vol 48no 5 pp 835ndash852 1976

[19] N Morales A Val del Rıo J R Vazquez-Padın R Mendez JL Campos and A Mosquera-Corral ldquoThe granular biomassproperties and the acclimation period affect the partial nitri-tationanammox process stability at a low temperature andammonium concentrationrdquo Process Biochemistry vol 51 no 12pp 2134ndash2142 2016

[20] R Blackburne Z Yuan and J Keller ldquoPartial nitrification tonitrite using low dissolved oxygen concentration as the mainselection factorrdquo Biodegradation vol 19 no 2 pp 303ndash3122008

[21] Y Ma Y Peng S Wang Z Yuan and X Wang ldquoAchievingnitrogen removal via nitrite in a pilot-scale continuous pre-denitrification plantrdquoWater Research vol 43 no 3 pp 563ndash5722009

[22] O Turk and D S Mavinic ldquoMaintaining nitrite build-up in asystem acclimated to free ammoniardquo Water Research vol 23no 11 pp 1383ndash1388 1989

[23] X Wang and D Gao ldquoIn-situ restoration of one-stage partialnitritation-anammox process deteriorated by nitrate build-upvia elevated substrate levelsrdquo Scientific Reports vol 6 Article ID37500 2016

[24] I Jubany J Lafuente J A Baeza and J Carrera ldquoTotal andstable washout of nitrite oxidizing bacteria from a nitrifyingcontinuous activated sludge system using automatic controlbased on Oxygen Uptake Rate measurementsrdquoWater Researchvol 43 no 11 pp 2761ndash2772 2009

[25] B Wett A Omari S M Podmirseg et al ldquoGoing for main-stream deammonification from bench to full scale for maxi-mized resource efficiencyrdquo Water Science and Technology vol68 no 2 pp 283ndash289 2013

[26] B Wett M Hell G Nyhuis T Puempel I Takacs and SMurthy ldquoSyntrophy of aerobic and anaerobic ammonia oxidis-ersrdquoWater Science and Technology vol 61 no 8 pp 1915ndash19222010

[27] X Li S Sun H Yuan B D Badgley and Z He ldquoMainstreamupflow nitritation-anammox systemwith hybrid anaerobic pre-treatment Long-term performance and microbial communitydynamicsrdquoWater Research vol 125 pp 298ndash308 2017

[28] T Kindaichi S Yuri N Ozaki and A Ohashi ldquoEcophysiologi-cal role and function of uncultured Chloroflexi in an anammoxreactorrdquoWater Science and Technology vol 66 no 12 pp 2556ndash2561 2012

[29] P Larsen J L Nielsen D Otzen and P H Nielsen ldquoAmyloid-like adhesins produced by floc-forming and filamentous bacte-ria in activated sludgerdquo Applied and Environmental Microbiol-ogy vol 74 no 5 pp 1517ndash1526 2008

[30] Z-R Chu K Wang X-K Li M-T Zhu L Yang and J ZhangldquoMicrobial characterization of aggregates within a one-stagenitritation-anammox system using high-throughput ampliconsequencingrdquo Chemical Engineering Journal vol 262 pp 41ndash482015

[31] M Fahrbach J Kuever RMeinke P Kampfer and J HollenderldquoDenitratisoma oestradiolicum gen nov sp nov a 17 120573-oestradiol-degrading denitrifying betaproteobacteriumrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 56 no 7 pp 1547ndash1552 2006

[32] B Kartal J Rattray L A van Niftrik et al ldquoCandidatusldquoAnammoxoglobus propionicusrdquo a new propionate oxidizingspecies of anaerobic ammonium oxidizing bacteriardquo Systematicand Applied Microbiology vol 30 no 1 pp 39ndash49 2007

Hindawiwwwhindawicom

International Journal of

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Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



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GenomicsInternational Journal of


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The Scientific World Journal

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BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


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Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


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Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


When treating mainstream wastewater that is character-izedwith low ammonium and ambient temperature stressingNOB and maintaining the activity of anammox bacteria arethe two key factors determining the CANON performance[14] Indeed substantial efforts have been made to optimizethe treatment process for a steady performance in CANONsystems when treatingmainstreamwastewater Han et al [15]used screens to separate flocs and granules in mainstreamdeammonification to wash out NOB and they found thatover 80 of NOB was washed out and up to 70 of nitrogenremoval efficiencies was achieved Similarly Malovanyy etal [14] tried integrated fixed film activated sludge reactorfor nitrogen removals from municipal wastewater using adeammonification process by intermittent aeration with ahighDO set-point (15mgL) and decreasing nonaerated time(30min) NOB was successfully out-selected However othereffective and operative approaches to ensure the effectivenitrogen removal performance of CANON systems are stillurgently required especially when treating low ammoniumwastewater

In this study a novel operational pattern for CANONsystem treating low ammonium wastewater was developedby alternatively inflowing low ammonium concentration(mimic mainstream wastewater) and high ammonium con-centration (mimic sidestream wastewater) The influentexchange frequency and the key controlling factors foreffective CANON operation were examined To elucidate themicrobial structure of functional bacteria and their influenceto the nitrogen removal performance the composition of thebacterial communities in the CANON system during oper-ational period was analyzed by 16S rDNA high-throughputsequencing technology The proposed operational strategyhopes to help CANON processes to be effectively applied inmunicipal wastewater treatment

2 Materials and Methods

21 Reactor Configuration A lab-scale sequencing batchreactor (SBR) with a working volume of 25 L was used in thisstudy The SBR was set in a thermostatic water bath to keeptemperature at 30∘CThe experimental biomass in the reactorwas mixed by a propeller stirrer at 100 rpm An aerationdevice linked with an air pump was fixed at the bottom of theSBR to provide oxygen for partial nitrification Air wasregularly supplied to the SBR by an air pump

22 Operational Strategies An alternative low and highammonium influent regime was adopted in the experimentAs shown in Figure 1 the whole experimental period wasdivided into four operational phases according to the influentammonium concentration Phase high I (0ndash60 d) Phase lowI (61ndash148 d) Phase high II (149ndash256 d) and Phase low II(257ndash285 d) Phases high I and II were operated at a highammonium concentration (approximately 240mgNH

4

+-NL) while Phases low I and II (approximately 61mgNH

4

+-NL) were operated at low ammonium concentration Atypical SBR cycle lasts for 4 h or 8 h respectively at low orhigh influent ammonium concentrations (Table 1) By oper-ating in an alternative low and high ammonium influent

regime the NOB inhibition and the dominance of AOB andanammox bacteria in the CANON system can be ensured

23 Seed Sludge and Feeding Medium The seeding granularsludge (25 L) for the experimental CANON systemwas fromanother CANON system treating high ammonium salinewastewater in our laboratory (Figure S1(A)) [3]

Synthetic wastewater was continuously introduced intothe reactor using a peristaltic pump in the inflow periodand the pump was controlled by a liquid level controller tocontrol the water volume of 25 L The composition of thesynthetic wastewater was KH

2PO4005 gL CaCl

203 gL

MgSO4sdot7H2O 03 gL NaHCO

3125 gL FeSO

4sdot7H2O

000625 gL Na2EDTA 000625 gL and 125mLL of trace

elements solution The composition of the trace elements so-lution was prepared according to Lotti et al [16] NH

4Cl was

added to the feeding medium to reach a desired ammoniumconcentration in the influent The pH was maintained at 80at the beginning of each cycle by adding hydrochloric acidsolution (02M) or sodium hydroxide solution (02M)

24 Analytical Methods Water samples were collected andstored in a 4∘C refrigerator until analysis The concentrationsof NH

4

+-N NO2

minus-N and NO3

minus-N were measured regularlyaccording to the standard methods [17] The DO concen-tration and pH were detected using online probes (WTWMulti350i Germany)

25 Sample Collection DNA Extraction and PCR Ampli-fication Sludge samples were collected from the CANONsystem on days 60 79 124 125 148 257 and 285 (the daysbefore the change in the operating condition) and stored atminus20∘Cafter centrifugation Total genomicDNAwas extractedin triplicate from each sample using the Power Soil DNAIsolationKit (Sangon China) according to themanufacturerrsquosinstructions The quality of the obtained genomic DNAwas examined by 1 (wv) agarose gel electrophoresis andconcentration measured with NanoDrop spectrophotometer2000 (Thermo Scientific USA)

Polymerase chain reaction amplification of the V3-V4region of the 16S rDNA gene was then conducted usingprimers 341F (51015840-CCTACACGACGCTCTTCCGATCTN-31015840)and 805R (51015840-GACTACHVGGGTATCTAATCC-31015840) with thereverse primer containing 6-bp barcodes to tag each sam-ple (Majorbio Bio-Pharm Technology Co Ltd ShanghaiChina) PCR amplification and 16S rDNA high-throughputsequencing were performed according to our previous study[3]

3 Results and Discussion

31 The Stabilization of the CANON System at High InfluentAmmonium Concentration The CANON system operatedfor 285 days and the long-term nitrogen removal per-formance was shown in Figure 1 The biomass was ini-tially cultivated with high ammonium concentration (196plusmn 18mgL) to set up the CANON system (Figure 1 Phasehigh I) After acclimation for 32 days the TN removal rate







Influent NH4




Aeration mode





0 50 100 150 200 250 3000

50

100

150

200

250

300

28525614860


Nitr

ogen

conc

entr

atio

n (m

gL)

Time (d)

Phase high I

00

01

02

03

04

05

TNRR

(kg

Nm

3 d)

TN

RE (

)

100

80

60

40

20

0

NH4+ inf

NO2minuseff

TNRR

NH4+eff

NO3minuseff

TNRE




4





0 10 20 30 40 50 60

0

5

10

15

20

25

Time (d)

In si

tu ac

tivity

(gN

m 3 d

)

00

01

02

03

04

05

06

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(a)

63 70 77 100 110 120 130 1400

5

10

15

20

25

30

Time (d)

02

04

06

08

10

In si

tu ac

tivity

(gN

m 3 h

)

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(b)

In si

tu ac

tivity

(gN

m 3 h

)

150 165 180 195 210 225 240 2550

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10Δ

NO3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(c)

In si

tu ac

tivity

(gN

m 3 d

)

260 265 270 275 280 2850

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(d)


minusΔNH4




4


4

+-N



4


4


3

minusΔNH4

+ increased




3

minusΔNH4


4




3

minusΔNH4

+



minusΔNH4



100 105 115 120 125 1304

6

8

10

12

14

16

Time (d)

y = 043x minus 379 (R2 = 098)

y = 001x minus 347 (R2 = 039)

NO

R (g

Nm

3d

)





3

minusΔNH4



3

minusΔNH4





minusΔNH4



3

minusΔNH4


3

minusΔNH4

+






4









100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(a)



100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(b)

0

10

20

30

40

50

60

C285C257C148C125C124C79

Relat

ive a

buda

nce (

)



C60Time (d)

(c)









4 Conclusions




Acknowledgments




3

minusΔNH4

+


References




































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








Influent NH4




Aeration mode





0 50 100 150 200 250 3000

50

100

150

200

250

300

28525614860


Nitr

ogen

conc

entr

atio

n (m

gL)

Time (d)

Phase high I

00

01

02

03

04

05

TNRR

(kg

Nm

3 d)

TN

RE (

)

100

80

60

40

20

0

NH4+ inf

NO2minuseff

TNRR

NH4+eff

NO3minuseff

TNRE




4





0 10 20 30 40 50 60

0

5

10

15

20

25

Time (d)

In si

tu ac

tivity

(gN

m 3 d

)

00

01

02

03

04

05

06

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(a)

63 70 77 100 110 120 130 1400

5

10

15

20

25

30

Time (d)

02

04

06

08

10

In si

tu ac

tivity

(gN

m 3 h

)

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(b)

In si

tu ac

tivity

(gN

m 3 h

)

150 165 180 195 210 225 240 2550

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10Δ

NO3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(c)

In si

tu ac

tivity

(gN

m 3 d

)

260 265 270 275 280 2850

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(d)


minusΔNH4




4


4

+-N



4


4


3

minusΔNH4

+ increased




3

minusΔNH4


4




3

minusΔNH4

+



minusΔNH4



100 105 115 120 125 1304

6

8

10

12

14

16

Time (d)

y = 043x minus 379 (R2 = 098)

y = 001x minus 347 (R2 = 039)

NO

R (g

Nm

3d

)





3

minusΔNH4



3

minusΔNH4





minusΔNH4



3

minusΔNH4


3

minusΔNH4

+






4









100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(a)



100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(b)

0

10

20

30

40

50

60

C285C257C148C125C124C79

Relat

ive a

buda

nce (

)



C60Time (d)

(c)









4 Conclusions




Acknowledgments




3

minusΔNH4

+


References




































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0 10 20 30 40 50 60

0

5

10

15

20

25

Time (d)

In si

tu ac

tivity

(gN

m 3 d

)

00

01

02

03

04

05

06

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(a)

63 70 77 100 110 120 130 1400

5

10

15

20

25

30

Time (d)

02

04

06

08

10

In si

tu ac

tivity

(gN

m 3 h

)

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(b)

In si

tu ac

tivity

(gN

m 3 h

)

150 165 180 195 210 225 240 2550

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10Δ

NO3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(c)

In si

tu ac

tivity

(gN

m 3 d

)

260 265 270 275 280 2850

5

10

15

20

25

30

Time (d)

00

02

04

06

08

10

ΔN

O3minusΔ

NH

4+

ΔNO3minusΔNH4

+

AOR NOR NRR

(d)


minusΔNH4




4


4

+-N



4


4


3

minusΔNH4

+ increased




3

minusΔNH4


4




3

minusΔNH4

+



minusΔNH4



100 105 115 120 125 1304

6

8

10

12

14

16

Time (d)

y = 043x minus 379 (R2 = 098)

y = 001x minus 347 (R2 = 039)

NO

R (g

Nm

3d

)





3

minusΔNH4



3

minusΔNH4





minusΔNH4



3

minusΔNH4


3

minusΔNH4

+






4









100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(a)



100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(b)

0

10

20

30

40

50

60

C285C257C148C125C124C79

Relat

ive a

buda

nce (

)



C60Time (d)

(c)









4 Conclusions




Acknowledgments




3

minusΔNH4

+


References




































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





3

minusΔNH4


4




3

minusΔNH4

+



minusΔNH4



100 105 115 120 125 1304

6

8

10

12

14

16

Time (d)

y = 043x minus 379 (R2 = 098)

y = 001x minus 347 (R2 = 039)

NO

R (g

Nm

3d

)





3

minusΔNH4



3

minusΔNH4





minusΔNH4



3

minusΔNH4


3

minusΔNH4

+






4









100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(a)



100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(b)

0

10

20

30

40

50

60

C285C257C148C125C124C79

Relat

ive a

buda

nce (

)



C60Time (d)

(c)









4 Conclusions




Acknowledgments




3

minusΔNH4

+


References




































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





minusΔNH4



3

minusΔNH4


3

minusΔNH4

+






4









100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(a)



100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(b)

0

10

20

30

40

50

60

C285C257C148C125C124C79

Relat

ive a

buda

nce (

)



C60Time (d)

(c)









4 Conclusions




Acknowledgments




3

minusΔNH4

+


References




































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(a)



100

80

60

40

20

0

Relat

ive a

bund

ance

()

C60 C79 C124 C125 C148 C257 C285Time (d)

(b)

0

10

20

30

40

50

60

C285C257C148C125C124C79

Relat

ive a

buda

nce (

)



C60Time (d)

(c)









4 Conclusions




Acknowledgments




3

minusΔNH4

+


References




































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









4 Conclusions




Acknowledgments




3

minusΔNH4

+


References




































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018
































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


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