decay experiments of effective n-removing microbial...

Research ArticleDecay Experiments of Effective N-Removing MicrobialCommunities in Sequencing Batch Reactors

Chen Lv12 Ming Li2 Shuang Zhong2 JianlongWang1 and Lei Wu2

1Laboratory of Environmental Technology Institute of Nuclear and New Energy Technology Tsinghua UniversityBeijing 100084 China2Key Laboratory of Songliao Aquatic Environment Ministry of Education Jilin Jianzhu University Changchun 130118 China

Correspondence should be addressed to Jianlong Wang wangjlmailtsinghuaeducn

Received 25 April 2017 Revised 27 July 2017 Accepted 14 August 2017 Published 14 September 2017

Academic Editor Claudio Di Iaconi

Copyright copy 2017 Chen Lv et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The temporal changes in the compositions of effective N-removing bacterial communities and the decay coefficients of Anammoxwere studied within the 120-day decay period under anaerobic or aerobic conditions at 25∘C The maximum nitrogen productionrate (MNPR) was determined bymeasuring the temperature pH volatile suspended solids (VSSs) and nitrogen-removal efficiencyof the microbial communities during the decay periodThe decay coefficients under anaerobic and aerobic conditions at 25∘Cweredetermined through equation-based fitting to be 0031 dminus1 and 0070 dminus1 respectively Through molecular biological means andtogether with quantitative polymerase chain reaction (qPCR) the proportions of AnAOB in the microbial communities droppedfrom 4870 to 369 under anaerobic condition and from 4870 to 198 under aerobic condition during the decay period

1 Introduction

Compared with traditional N-removal processes the Anam-mox process is superior with low investment and operationcosts low sludge yield high processing efficiency and feasi-bility to wastewater with low CN ratio and high ammonianitrogen [1] However Anammox is limited by extremely lowcell yield slow cell growth environmental sensitivity andhigh requirements for temperature pH water and substrateduring cultivation [2 3] Moreover the Anammox process islimited by the difficulty in starting instability after start-upand difficulty in recovery after destabilization [3ndash5] Theseproblems can be overcome if there are abundant favorablebacterial species that can be used for early-phase inoculationor anaphase fed-batch [6] In current activated sludgemodelsof aerobic degradation the loss of activity and mass ofactivated sludge is expressed by only one process calleddecay [7] And the decrease in bacterial activity in activatedsludge can result from cell death and activity decay [8] andsignificantly affects the preservation of bacterial species andthe decay coefficient [9 10] Moreover the decay coefficientis one of the main variables in the mathematical modeling

that is applied to biological wastewater processing Correctestimation of the decay constant is a key factor to properlymodel and better understand the Anammox process it isalso a parameter that is helpful to design and manage anAnammox reactor [11]

The objectives of this study are to accurately measure thedecay coefficient of N-removing functional microbes duringthe whole decay period and together with quantitative poly-merase chain reaction (qPCR) to analyze the compositionalchanges of functional microbial communities which weremaintained under anaerobicaerobic conditions and withoutfeeding for about 4 months

2 Materials and Methods

21 Materials All the microbe samples in the decay experi-ments were collected from a laboratory small-scale sequenc-ing batch reactor (SBR) fermentation tank (4 L) The reactorran under controlled conditions (30∘C pH 75 plusmn 05) and wasblended and stirred at the speed of 80 rpm by the machinerystirrer in the fermentation tank The reactor had a runningperiod of 8 h drainage ratio of 50 hydraulic retention time

HindawiJournal of ChemistryVolume 2017 Article ID 4878910 5 pageshttpsdoiorg10115520174878910

2 Journal of Chemistry

(HRT) of 16 h and volume nitrogen load rate of 750mgNLsdotdOther devices included a temperature control set installedoutside the fermentation tank the annular aeration line atthe bottom and an online data monitor for measurement ofdissolved oxygen (DO) pH temperature NH4

+ and NO3minus

The composition of the inflowwaterwas 1697mgLKH2PO47511mgLMgSO4sdot7H2O 4516mgL CaCl2sdot2H2O 200mgLEDTA 500mgL FeSO4sdot7H2O 043mgL ZnSO4sdot7H2O024mgL CoCl2sdot6H2O 099mgL MnCl2sdot4H2O 025mgLCuSO4sdot5H2O 022mgL NaMoO4sdot2H2O 019mgLNiCl2sdot6H2O and 021mgL NaSeO4sdot10H2O22 Experimental Methods The decay experiments of N-removing functional microbial communities were conductedunder anaerobic or aerobic conditions at 25∘C Four groupswere conducted each in triplicate The microbial communi-ties taken out of the reactor were washed with the substrate-free inflow water Each time 250mL of a sample was placedinto a 300mL sealed bottle which was put under thecorresponding experimental condition At a certain interval25mL of the sample was collected pretreated and measuredin terms of NH4

+NO2minusNO3

minus pH volatile suspended solids(VSS) specific Anammox activity (SAA) and qPCR Thedecay changes of N-removing microbial communities underdifferent conditions were described quantitatively

SAA was detected using the method from Buys [12] Thismethod was first applied into denitrifying bacteria but itwas also feasible to measurement of low biomass and lowgas production so it was used in this study The principle ofbacterial activity test is that an appropriate ratio of NH4

+ andNO2minus was added to the substrate and then N2 production

was detected The anaerobic condition was realized by theventilation of nitrogen gas into the reactor Under the anaer-obic condition the substrate (NH4

+ andNO2minus each 5mmol)

was added According to (1) if the functional microbialcommunity was active the generated gas should be N2

NH4+ + 132NO2minus + 0066HCO3

minus + 013H+ 997888rarr102N2 + 0256NO3minus + 0066CH2O05N015 + 203H2O

(1)

SAA can be used tomeasure the newly added air pressurein the test bottle (unit mV) or namely the N2 productionfrom the reaction Then we could determine the nitrogenproduction rate 119899 as follows

119875119881 = 119899119877119879 (2)

where 119875 is the overpressure (higher than normal pressure)119881 is the space volume in the top 119877 is the ideal gas constant(=00821 atmsdotmLKsdotmmol) 119879 is the temperature

According to (2) we could determine the maximumnitrogen production rate (MNPR) which is proportional by119870 to the biomass solid concentration119883anx(119905) Thus we couldestimate the decay coefficient 119887AN as follows

MNPR (119905) = 119896 lowast 119883anx (119905)119889119909anx119889119905 = minus119887AN lowast 119883anx119883anx (119905) = 119883anx(119905=0)119890minus119887ANlowast119905

(3)

5 10 15 20 250Time (hour)

000

005

010

015

020

025

mm

ol-N

（4+

2minus 2

3minus

Figure 1 Initial N-removing performance of functional microbialcommunities

23 Analytical Methods VSS was measured by a standardmethod pH was measured by a PHM210 device N-con-taining particle concentration was detected by a colorimetrickit after filtration by a 045 120583m acetic acid fibrin injector(Merck KGaA Darmstadt Germany)

In DNA extraction a microbe sample after frozen dryingat minus50∘C was weighed and total DNA from each activatedsludge sample was extracted using an MP soil DNA rapidextraction kit (Bio101 Vista CA USA) according to themanualThe qPCR amplification of 16rRNA functional geneswas conducted with the following primers 1055f1391r (EUB)[13 14] CTO 189fABRT1r (AOB) [15] Nspra-675f746r(NOB) [16] and Amx809f1066r (AnAOB) [17 18]

3 Results and Discussion

31 Initial N-Removing Performances of Functional MicrobialCommunities The initial activity of each functional micro-bial community was measuredThe results were atmosphericpressure = 18mV and initial MNPR = 263mLN2-NLsdotd Theair pressure peaked within 24 h while the N2 productionduring the whole reaction increased with time which provesthe initial microbes were highly active

The N2 production was measured simultaneously withsampling The measurements of N-containing particles wereconverted to nitrogen molar concentrations (Figure 1)Clearly at the 16th hour under the action of the functionalmicrobial communities the newly added NH4

+ and NO2minus

(totally 03mmol-N) almost all reacted forming 004mmol-N NO3

minus and 025mmol-N N2 and the change of molarconcentrations obeyed (1) which proves that the initial func-tional microbial communities had high N-removing ability

32 Variation of VSS with Time During the 120-day decayperiod the functional microbial communities were sampledand it was found the VSSs significantly declined under all testconditions (Figure 2) Under the anaerobic condition VSSsdropped at a significant rate whichwas slower than under theaerobic conditionTheVSSs under the anaerobic condition in

Journal of Chemistry 3

Oct Nov Dec JanSepTime (month)

00

02

04

06

08

10

12

VSS

(gVS

SL)

Anaerobic 25∘CAerobic 25∘C

Figure 2 Variation of VSS during 120 days

Oct Nov Dec and Jan were 082 077 064 and 048 gVSSLrespectively Under the aerobic condition the VSS in Oct didnot change significantly and was 103 gVSSL Neverthelessthe color changed significantly and turned light grey whichindirectly indicates the reduction of microbial activity TheVSS in Nov declined severely from the previous month andwas 038 gVSSL The VSSs in Dec and Jan were 023 and024 gVSSL respectively indicating the microbes almost alldecayed in the third and fourth months

33 Variation of MNPR with Time In a reactor undercontinuous stirring the equation derived from the massbalance under stable conditions represents the relationshipbetween119883AN and 119887AN119883AN = 119884AN sdot (NH4

+IN minusNH4+ OUT) sdot SRT

HRT times (1 + 119887AN sdot SRT)SRT 997888rarr infin

119883AN 997888rarr 119884AN sdot (NH4+

IN minus NH4+ OUT)HRT times 119887AN

(4)

Thuswhen 119887AN and other bioreactor parameters [hydrau-lic retention time (HRT) sludge retention time (SRT) nitro-gen-removal efficiency and nitrogen load] are known theAnammox biomass concentration can be easily estimated[11] In the following equation the relevant parameters werecited from an Anammox reaction equation [19] and theMNPR (mLN2-NLsdotd) was associated with active biomass(119883AN)

MNPR (119905) = (120583max sdot 204 sdot 224119884AN sdot 14 sdot 2 ) sdot 119883ANX (119905)

= 119896 sdot 119883ANX (119905 = 0) sdot 119890minus119887AN sdot119905(5)

where 119887AN is the decay coefficient (dminus1) 120583max is themaximumgrowth rate (dminus1) 119883AN is the concentration of Anammoxorganisms (mgCODLminus1) 119884AN is the Anammox growth yield


00

05

10

15

20

25

30

ml

2-N

Lmiddotd

)M

NPR

(


Figure 3 Variation of MNPR with time

(mgCODmgNH4-Nminus1) and 119896 is the maximum specific

nitrogen gas production rate (mLN2-N Lminus1 dminus1mgCODminus1)The SAAs during the 120-day decay period were mea-

sured and used to determine the MNPR according to (5)As shown in Figure 3 the MNPR (mLN2-NLsdotd) is 263 atfirst and then declines significantly with time Under theanaerobic condition the MNPRs in Oct Nov Dec and Janare 095 05 03 and 02mLN2-NLsdotd respectively Underthe aerobic condition MNPRs decline significantly and are025 027 and 026mLN2-NLsdotd in the first three monthsrespectively In the fourthmonth nearly no air pressure couldbe detected which indicates the inactivity of the functionalmicrobial communityThe above results suggest that MNPRsunder the anaerobic condition decline regularly and in agradient way with the prolonging of time MNPR under theaerobic condition drops rapidly in the first month but doesnot change severely in the following three months indicatingthat theO2 concentration largely affects the decaying process

34 Decay Coefficient 119887AN of Functional Microbes The decaycoefficient (119887AN) is commonly used in mathematical model-ing of biological wastewater processing Specifically for themajority of Anammox process reactors the balanced con-centration of active biomass is largely dependent on 119887AN119887AN can be determined by fitting NMPR according to (5)on software (Figure 4) 119887AN of themicrobes under the anaero-bic and aerobic conditions at 25∘C are 0031 dminus1 and 0070 dminus1respectively indicating at the same temperature 119887AN underthe aerobic condition is two times larger than the anaerobiccondition Strous et al studied the effects of oxygen onAnammox by using SBR [19] In a reactor the anaerobic andaerobic conditions were alternated and Anammox reactionoccurred only after the stop of oxygen supply but not duringoxygen supply Thus the experiments prove that oxygensupply could inhibit the activity of Anammox which canbe restored after oxygen supply Nevertheless the inhibitoryeffect of oxygen concentrations on the Anammox activityshould be further studied When the oxygen concentrationwas 05ndash20 of the saturated oxygen concentration in air


0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6264eminus0031x

R2 = 09942

0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6302eminus0070x

R2 = 09719

ml

2-N

Lmiddotd

)M

NPR

( ml

2-N

Lmiddotd

)M

NPR

( Exponential decacy model of anaerobic 25∘C Exponential decacy model of aerobic 25∘C

Figure 4 Fitting curves of NMPR during the decay period under different conditions


0

20

40

60

80

100

()

AnAOBAOB

NOBothers

Anaerobic 25∘C

Figure 5 Proportion variation of species in the total functionalmicrobial communities under anaerobic condition

the activity of Anammoxwas completely inhibited indicatingthat the inhibitory oxygen concentration on the activity ofAnammox is 05 of saturated oxygen concentration inair Through our experiments we not only determined thedecay coefficient but also validated that again the oxygenconcentration could inhibit the activity of Anammox

35 Molecular Biology Analysis Fluorescence qPCR can beused to detect the copy number of a gene in an unknownsample Namely a known concentration of the gene wasdiluted to a series of gradient concentrations which weredetected through one qPCR trial The results determinedfrom this gradient of concentrations were used to plot astandard curve from which we could deduce the con-centration of an unknown sample The detected microbeswere divided into four classes AOB NOB AnAOB andothers Based on qPCR the measured data were used toestimate the proportion of each bacterial species in the overallbiomass When the proportion declines the decaying speed

0

20

40

60

80

100

()

AnAOBAOB

NOBothers


Anaerobic 25∘C

Figure 6 Proportion variation of species in the total functionalmicrobial communities under aerobic condition

of this species surpasses the average rate of other speciesand vice versa The functional microbial communities wereinitially composed of AOB (1080) AnAOB (4870) NOB(087) and others (3963)

Under the anaerobic condition the functional micro-bial communities consisted of AnAOB (3050) and others(6037) (Oct) AnAOB (1570) and others (7686) (Nov)AnAOB (404) and others (8844) (Dec) AnAOB (392)and others (9053) (Jan) (Figure 5) Under the aerobiccondition the functionalmicrobial communities consisted ofAnAOB (398) and others (9631) (Oct) AnAOB (298)and others (8951) (Nov) AnAOB (198) and others(9080) (Dec) AnAOB (230) and others (9179) (Jan)(Figure 6) Clearly under the aerobic condition the decayingrate of AnAOB surpassed those of other species throughoutthe experiments indicating that oxygen is extremely unfa-vorable for the survival of AnAOB and the decay of AnAOBis severe under aerobic conditions The results of qPCR areconsistent with the results of VSS MNPR and 119887AN


4 Conclusions

Correct evaluation of decay coefficient helps to better under-stand the Anammox process The decay of functional micro-bial communities in 120-day experiments was monitoredMNPR was determined by measuring the temperature pHVSS and nitrogen-removal efficiency of microbial commu-nities during the decay period The decay coefficients underanaerobic and aerobic conditions at 25∘C were determinedthrough fitting to be 0031 dminus1 and 0070 dminus1 respectively

Throughmolecular biologymeans AnAOB communitiesdecreased faster under aerobic than anaerobic conditionThe proportion of AnAOB in the microbial community isproportional to the N-removing efficiency

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] S Tomar and S K Gupta ldquoInvestigating the role of co-sub-strate-substrate ratio and filter media on the performance ofanammox hybrid reactor treating nitrogen rich wastewaterrdquoJournal of Bioscience and Bioengineering vol 121 no 3 pp 310ndash316 2016

[2] Z Lei andZ Ping ldquoMetabolismof anaerobic ammoniumoxida-tion (anammox) bacteriardquo Bulletin of Science Technology andSociety vol 26 no 6 pp 931ndash937 2010

[3] W Cai-hua Z Ping C Jing and C Ting-ting ldquoPreservationof ANAMMOX bacteriardquo China Environmental Science vol 33no 8 pp 1474ndash1482 2013

[4] AGMutlu A K Vangsgaard G Sin and B F Smets ldquoAn oper-ational protocol for facilitating start-up of single-stage auto-trophic nitrogen-removing reactors based on process stoi-chiometryrdquoWater Science andTechnology vol 68 no 3 pp 514ndash521 2013

[5] B-S Xing Q Guo X-Y Jiang et al ldquoInfluence of preservationtemperature on the characteristics of anaerobic ammoniumoxidation (anammox) granular sludgerdquo Applied Microbiologyand Biotechnology vol 100 no 10 pp 4637ndash4649 2016

[6] W Cai-hua Z Ping C Jing and C Ting-ting ldquoEffectsof intermittent starvation on preservation characteristics ofANAMMOX bacteriardquo Acta Scientiae Circumstantiae vol 33no 1 pp 36ndash43 2013

[7] M Friedrich and I Takacs ldquoA new interpretation of endoge-nous respiration profiles for the evaluation of the endogenousdecay rate of heterotrophic biomass in activated sludgerdquoWaterResearch vol 47 no 15 pp 5639ndash5646 2013

[8] X Hao Q Wang X Zhang Y Cao and C M V MarkLoosdrecht ldquoExperimental evaluation of decrease in bacterialactivity due to cell death and activity decay in activated sludgerdquoWater Research vol 43 no 14 pp 3604ndash3612 2009

[9] XHaoQWang YCao andMCMVanLoosdrecht ldquoExperi-mental evaluation of decrease in the activities of polyphos-phateglycogen-accumulating organisms due to cell death andactivity decay in activated sludgerdquo Biotechnology and Bioengi-neering vol 106 no 3 pp 399ndash407 2010

[10] R Manser W Gujer and H Siegrist ldquoDecay processes ofnitrifying bacteria in biological wastewater treatment systemsrdquoWater Research vol 40 no 12 pp 2416ndash2426 2006

[11] D Scaglione S Caffaz E Bettazzi and C Lubello ldquoExperimen-tal determination of Anammox decay coefficientrdquo Journal ofChemical Technology and Biotechnology vol 84 no 8 pp 1250ndash1254 2009

[12] B R Buys A Mosquera-Corral M Sanchez and R MendezldquoDevelopment and application of a denitrification test based ongas productionrdquoWater Science and Technology pp 41-12 2000

[13] H Qianyi Z Ping and K Da ldquoTaxonomy characteristics andbiotechniques used for the analysis of anaerobic ammoniumoxidation bacteriardquoChinese Journal of Applied and Environmen-tal Biology vol 02 pp 384ndash391 2017

[14] W Yinhua M A Yuexin L Changfa Z Xuehui and Z YingldquoThe bacterial diversity in boifilms associated with media ofanammox bioreactorsrdquo Journal of Ocean University of Chinavol 26 no 6 pp 500ndash506 2011

[15] P Fangfang Z Peng M Hang L Kaihong and P Yang-fang ldquoComparing the primer specificity for betaproteobacterialammonia-oxidizing bacteria in recirculation aquaculture sys-temsrdquo Acta Microbiologica Sinica vol 51 no 10 pp 1342ndash13502011

[16] T Katipoglu-Yazan C Merlin M-N Pons E Ubay-Cokgorand D Orhon ldquoChronic impact of sulfamethoxazole on themetabolic activity and composition of enriched nitrifyingmicrobial culturerdquoWater Research vol 100 pp 546ndash555 2016

[17] A Daverey S-H Su Y-T Huang S-S Chen S Sung and J-GLin ldquoPartial nitrification and anammox process A method forhigh strength optoelectronic industrial wastewater treatmentrdquoWater Research vol 47 no 9 pp 2929ndash2937 2013

[18] A Daverey Y-C Chen K Dutta Y-T Huang and J-G LinldquoStart-up of simultaneous partial nitrification anammox anddenitrification (SNAD) process in sequencing batch biofilmreactor using novel biomass carriersrdquo Bioresource Technologyvol 190 pp 480ndash486 2015

[19] M Strous J J Heijnen J G Kuenen and M S M Jetten ldquoThesequencing batch reactor as a powerful tool for the study ofslowly growing anaerobic ammonium-oxidizing microorgan-ismsrdquoAppliedMicrobiology and Biotechnology vol 50 no 5 pp589ndash596 1998

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Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


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Medicinal ChemistryInternational Journal of


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Applied ChemistryJournal of



Theoretical ChemistryJournal of


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Spectroscopy

Analytical ChemistryInternational Journal of


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Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


(HRT) of 16 h and volume nitrogen load rate of 750mgNLsdotdOther devices included a temperature control set installedoutside the fermentation tank the annular aeration line atthe bottom and an online data monitor for measurement ofdissolved oxygen (DO) pH temperature NH4

+ and NO3minus

The composition of the inflowwaterwas 1697mgLKH2PO47511mgLMgSO4sdot7H2O 4516mgL CaCl2sdot2H2O 200mgLEDTA 500mgL FeSO4sdot7H2O 043mgL ZnSO4sdot7H2O024mgL CoCl2sdot6H2O 099mgL MnCl2sdot4H2O 025mgLCuSO4sdot5H2O 022mgL NaMoO4sdot2H2O 019mgLNiCl2sdot6H2O and 021mgL NaSeO4sdot10H2O22 Experimental Methods The decay experiments of N-removing functional microbial communities were conductedunder anaerobic or aerobic conditions at 25∘C Four groupswere conducted each in triplicate The microbial communi-ties taken out of the reactor were washed with the substrate-free inflow water Each time 250mL of a sample was placedinto a 300mL sealed bottle which was put under thecorresponding experimental condition At a certain interval25mL of the sample was collected pretreated and measuredin terms of NH4

+NO2minusNO3

minus pH volatile suspended solids(VSS) specific Anammox activity (SAA) and qPCR Thedecay changes of N-removing microbial communities underdifferent conditions were described quantitatively

SAA was detected using the method from Buys [12] Thismethod was first applied into denitrifying bacteria but itwas also feasible to measurement of low biomass and lowgas production so it was used in this study The principle ofbacterial activity test is that an appropriate ratio of NH4

+ andNO2minus was added to the substrate and then N2 production

was detected The anaerobic condition was realized by theventilation of nitrogen gas into the reactor Under the anaer-obic condition the substrate (NH4

+ andNO2minus each 5mmol)

was added According to (1) if the functional microbialcommunity was active the generated gas should be N2

NH4+ + 132NO2minus + 0066HCO3

minus + 013H+ 997888rarr102N2 + 0256NO3minus + 0066CH2O05N015 + 203H2O

(1)

SAA can be used tomeasure the newly added air pressurein the test bottle (unit mV) or namely the N2 productionfrom the reaction Then we could determine the nitrogenproduction rate 119899 as follows

119875119881 = 119899119877119879 (2)

where 119875 is the overpressure (higher than normal pressure)119881 is the space volume in the top 119877 is the ideal gas constant(=00821 atmsdotmLKsdotmmol) 119879 is the temperature

According to (2) we could determine the maximumnitrogen production rate (MNPR) which is proportional by119870 to the biomass solid concentration119883anx(119905) Thus we couldestimate the decay coefficient 119887AN as follows

MNPR (119905) = 119896 lowast 119883anx (119905)119889119909anx119889119905 = minus119887AN lowast 119883anx119883anx (119905) = 119883anx(119905=0)119890minus119887ANlowast119905

(3)

5 10 15 20 250Time (hour)

000

005

010

015

020

025

mm

ol-N

（4+

2minus 2

3minus

Figure 1 Initial N-removing performance of functional microbialcommunities

23 Analytical Methods VSS was measured by a standardmethod pH was measured by a PHM210 device N-con-taining particle concentration was detected by a colorimetrickit after filtration by a 045 120583m acetic acid fibrin injector(Merck KGaA Darmstadt Germany)

In DNA extraction a microbe sample after frozen dryingat minus50∘C was weighed and total DNA from each activatedsludge sample was extracted using an MP soil DNA rapidextraction kit (Bio101 Vista CA USA) according to themanualThe qPCR amplification of 16rRNA functional geneswas conducted with the following primers 1055f1391r (EUB)[13 14] CTO 189fABRT1r (AOB) [15] Nspra-675f746r(NOB) [16] and Amx809f1066r (AnAOB) [17 18]

3 Results and Discussion

31 Initial N-Removing Performances of Functional MicrobialCommunities The initial activity of each functional micro-bial community was measuredThe results were atmosphericpressure = 18mV and initial MNPR = 263mLN2-NLsdotd Theair pressure peaked within 24 h while the N2 productionduring the whole reaction increased with time which provesthe initial microbes were highly active

The N2 production was measured simultaneously withsampling The measurements of N-containing particles wereconverted to nitrogen molar concentrations (Figure 1)Clearly at the 16th hour under the action of the functionalmicrobial communities the newly added NH4

+ and NO2minus

(totally 03mmol-N) almost all reacted forming 004mmol-N NO3

minus and 025mmol-N N2 and the change of molarconcentrations obeyed (1) which proves that the initial func-tional microbial communities had high N-removing ability

32 Variation of VSS with Time During the 120-day decayperiod the functional microbial communities were sampledand it was found the VSSs significantly declined under all testconditions (Figure 2) Under the anaerobic condition VSSsdropped at a significant rate whichwas slower than under theaerobic conditionTheVSSs under the anaerobic condition in



00

02

04

06

08

10

12

VSS

(gVS

SL)







119883AN 997888rarr 119884AN sdot (NH4+


(4)






00

05

10

15

20

25

30

ml

2-N

Lmiddotd

)M

NPR

(








0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6264eminus0031x

R2 = 09942

0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6302eminus0070x

R2 = 09719

ml

2-N

Lmiddotd

)M

NPR

( ml

2-N

Lmiddotd

)M

NPR




0

20

40

60

80

100

()

AnAOBAOB

NOBothers

Anaerobic 25∘C




0

20

40

60

80

100

()

AnAOBAOB

NOBothers


Anaerobic 25∘C





4 Conclusions





References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



00

02

04

06

08

10

12

VSS

(gVS

SL)







119883AN 997888rarr 119884AN sdot (NH4+


(4)






00

05

10

15

20

25

30

ml

2-N

Lmiddotd

)M

NPR

(








0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6264eminus0031x

R2 = 09942

0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6302eminus0070x

R2 = 09719

ml

2-N

Lmiddotd

)M

NPR

( ml

2-N

Lmiddotd

)M

NPR




0

20

40

60

80

100

()

AnAOBAOB

NOBothers

Anaerobic 25∘C




0

20

40

60

80

100

()

AnAOBAOB

NOBothers


Anaerobic 25∘C





4 Conclusions





References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6264eminus0031x

R2 = 09942

0

10

20

30

40

50

60

70

20 40 60 80 100 1200Time (day)

y = 6302eminus0070x

R2 = 09719

ml

2-N

Lmiddotd

)M

NPR

( ml

2-N

Lmiddotd

)M

NPR




0

20

40

60

80

100

()

AnAOBAOB

NOBothers

Anaerobic 25∘C




0

20

40

60

80

100

()

AnAOBAOB

NOBothers


Anaerobic 25∘C





4 Conclusions





References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


4 Conclusions





References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

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decay experiments of effective n-removing microbial...

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