Influence of Temperature on MBBR Denitrification for Advanced Nitrogen Removal

of Wastewater Treatment Plant EffluentHaiyan Wang

K. Liu,Q.Y. Hang, Q. Yuan, M.J. Ma, and C. M. Li

Research Center for Water Pollution Control Technology

Chinese Research Academy of Enviromental Sciences

2016.09.16Tel:86-10-84915322, Email: wanghy@craes.org.cn

Results and DisscussionResults and Disscussion44Materials and MethodsMaterials and Methods33

Outline

BackgroundBackground11

ConclusionsConclusions55

ObjectiveObjective22

BACKGROUND1

Advanced nitrogen removal is necessary for the WWTP effluent reuse

1• Water shortage has becoming a serious problem • Water shortage has becoming a serious problem 

2

• The amount of municipal wastewater treatment plant (WWTP) effluent was large(3.4 × 1010 ton/year)

• WWTP effluent is a good resource as reclaimed water.

• The amount of municipal wastewater treatment plant (WWTP) effluent was large(3.4 × 1010 ton/year)

• WWTP effluent is a good resource as reclaimed water.

3 •TN 15mg/L ？ 1.5mg/L•TN 15mg/L ？ 1.5mg/L80.8±8.4% NO3

--N

Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant (GB18918-2002)

Environmental Quality Standards for Surface Water(GB3838-2002)

IV recharge water for water sources,natural wetlands or rivers

Moving Bed Biofilm Reactor

ActivedSludge

Bioflim

MBBR

Influent

Aeration/Mixing

Effluent

Denitrification MBBR reported for sewage wastewater (Aspegren et al., 1998; 

Mases et al. 2010), nitrate contaminated seawater(Labelle, et al., 2005) and 

landfill leachate (Cortez et al., 2011) nitrogen removal with good 

denitrification effficiency.

Denitrifiction MBBR could be used for WWTP effluent reuse

The full‐scale MBBRs had also been applied since 1997 & 1999 for post denitrification in Sjölunda and Klagshamn WWTPs of Malmö City, Sweden, and the effluent TN could met the WWTP limitation (Mases et al. 2010).

Influencing factors temperature

the ratio of carbon to total nitrogen(C/N)

mixing speed

Carriers types

hydraulic retention time (HRT)

OBJECTIVE2

Objective

• The denitrification MBBR has been developed  for WWTP effluent advanced nitrogen removal, and the influencing of carriers types, hydraulic retention time (HRT) and C/N ratio on its efficiency have been studied extensively in our published studies (Yuan et al. 2015; 2016). 

Temperatureinfluence

Microorganisms 

WWTPs built outside

Variation obviously in different seasons in North China

denitrification efficiency HOW the denitrificans community change?

Denitrification functional Genes

nosZ

N2O N2

Nar

narG

napA

Nir

nirS

nirK

Nor

norB

norZ

Nap Cu‐Nir qNor

Nos

NONO3- NO2

-

qPCR molecular approach

MATERAIALS AND METHODS

3

Equipments and carriers

MBBR : 6.0 L volume and 5.7 L effective volumeInfluent : Secondary sedimentation tank effluent of Beijing Yongfeng WWTPC/N ratio: methanol addition to adjust C/N ratio to 8 HRT 12h

Polyethylene carriers with 25 mm diameter, 10 mm height, 0.96~0.98 g/L density and 620 m2/m3 specific surface area.

Time(d) Temperature( )  CODCr(mg/L)  TN(mg/L) NO3‐‐N (mg/L)

Phase I(1‐49d) 13 104.6±86.3 11.1±7.0 4.8±2.6

PhaseII(50‐98d) 19 109.7±82.0 12.0±7.6 4.2±2.6

PhaseIII(99‐147d) 25 87.8±29.7 10.7±2.7 3.9±2.1

PhaseIV(148‐190d) 30 85.6±30.4 10.6±2.7 4.0±2.1

Table1 Influent ingredients and operation conditions

PE

Sequencing batch test in one HRT

Time(d) Temperature()  CODCr(mg/L)  TN(mg/L) NO3

‐‐N (mg/L)

1‐3 30 117.8 11.1 4.6

4‐6 25 109.0 11.9 4.1

7‐9 20 91.4 11.8 4.5

10‐12 15 113.4 11.2 4.3

13‐15 10 95.8 11.6 4.4

MBBR : 2.5 L volume and 2.0 L effective volumeInfluent : Secondary sedimentation tank effluent of Beijing Yongfeng WWTPCarriers filling rate: 30% C/N ratio: methanol  addition to adjust C/N ratio to  8 HRT:  12hSampling once every hour for 8 times in one HRT

Analytical methods

TN :TN unit of TOC‐VCPH analyzerNO3

‐‐N and NO2‐‐: Ion chromatography (Dionex ICS‐1000

NH4+‐N :Nessler's reagent spectrophotometry method

COD :COD speedy testing pH :S‐25 pH Analyzer

Water Quality Index Detection

DOM analysisThree‐dimensional excitation‐emission matrix (EEM) fluorescence spectrophotometerExcitation source was used for the spectrometer, both excitation (Ex) and emission (Em) were 200‐450 nm with 5 nm bandwidth, and the scanning speed was 12000 nm•min‐1.

Analytical methods

DNA extraction and qPCR analysisDNA extraction: UltraClean DNA extraction kitTwo‐step Q‐PCR amplification procedure: 95 for 10 min; 40 cycles of 95 for 15 s, 60 for 1 min. 

Primers References

NarG‐F  5’‐TA(CT)GT(GC)GGGCAGGA(AG)AAA ‐3’ Smith et al., 2015

NarG‐R 5’‐ CGTAGAAGAAGCTGGTGCTGTT‐3’ 

NirS‐F 5’‐ GTSAACGTSAAGGARACSGG‐3’  Kandeler et al., 2006

NirS‐R 5’‐ GASTTCGGRTGSGTCTTGA‐3’ NosZ‐F  5’‐ CG(C/T)TGTTC(A/C)TCGACAGCCAG ‐3’ Mao et al., 2011

NosZ‐R 5’‐ G(G/C)ACCTT(G/C)TTGCC(C/G)T(T/C)GCG ‐3

Biomass analysis and SEM observation

Biomass: SS weight method (Liu, 1992)SEM:HITACHI S‐570 SEM

RESULTS AND DISCUSSION4

Influence of  temperature on Nitrogen Removal

Influence of temperature on NO3‐‐N Removal

0 50 100 150 2000

4

8

12

16

20

0

20

40

60

80

100

（ ）Time d Influent Effluent Removal rate

30( )25( )19( )13( )

NO

- 3-N r

emov

al ra

te(%

)

NO

- 3-N(m

g/L)

The NO3--N·removal and denitrification rate changed little with the temperature variation,

and it indicated the temperature has little effect on the nitrate removal at 12h HRT, which might be because the long HRT (12h) of the MBBR could compensate for the low denitrification rate at low temperature.

Temperature( )  NO3

‐‐N (mg/L) NO3—N removal(%)

NO3‐‐N removal 

rate (mg NO3‐‐

N∙(L∙d)‐1)13 4.8±2.6 82.9±26.7 %,  4.8±2.6

19 4.2±2.6 80.1±27.2 % 4.2±2.6

25 3.9±2.1 85.0±22.3 % 3.9±2.1

30 4.0±2.1 82.9±13.3% 4.0±2.1

Influence of  temperature on Nitrogen Removal

Influence of temperature on NO3‐‐N Removal 

in sequencing batch tests

-1 0 1 2 3 4 5 6 7 8 9

0

1

2

3

4

5

NO

- 3-N/(m

g/L)

Time(h)

10( ) 15( ) 20( ) 25( ) 30( )

Temperature(

) 

Time for complete NO3

—N removal (h)

The denitrificationRate(mg/L∙h)

10 7 0.64

15 7 0.58

20 6 1.12

25 5 1.29

30 5 1.14

With the long HRT(12h), the microorganisms at low temperature could arrive the same removal efficiency as that of the high temperature for the time compensation (Zinatizadeh & Ghaytooli, 2015). The batch tests also explained why the similar nitrate removal were obtained at long HRT (12h ) at 13 , 19 , 25 and 30 .

Influence of  temperature on  the Removal of Nitrogen in Other Form Temperatu

‐re ( )NH4

+‐N NO2‐‐N TN

Influent (mg/L)

Effluent(mg/L) Removal Influent

(mg/L)Effluent(mg/L) Removal Influent

(mg/L)Effluent(mg/L) Removal

13 0.8±0.7 0.5±0.4 27.2±46.3 2.0±1.2 1.8±0.8 4.3±3.6 11.1±6.9 5.5±4.

153.1±28.4

19 0.9±0.8 0.7±0.4 23.5±51.1 2.0±1.2 1.9±0.7 4.1±3.3 12.0±6.9 6.2±4.

249.0±28.2

25 0.8±0.3 0.6±0.4 30.5±24.9 2.3±0.7 2.3±0.6 4.2±7.1 10.7±2.7 4.9±2.

252.8±16.8

30 0.7±0.4 0.5±0.3 31.3±11.1 2.5±0.6 2.4±0.7 6.1±7.3 10.5±2.8 4.8±3.

054.6±23.9

The low NH4+-N and NO2

--N varied little with the temperature, and they were not influenced obviously by the temperature in the batch tests. TN were also not affected by temperature obviously.

Influence of  temperature on Nitrogen Removal

Influence of  temperature on Organic Removal

COD removal

The CODCr removal were 53.3±44.9%, 52.5±30.6%, 65.5±18.6% and 63.8±19.5%, which indicated that the temperature had little effect on the COD removal

In batch tests, the CODcr removal were steady after 5h operation, and the CODCr removal rate increased gradually with the temperatures of 10 , 15 , 20 , 25 and 30 .

Influence of  temperature on Organic Removal

DOM removal

Effluent

The total fluorescence intensity removal were 47.6%, 49.0%, 50.5% and 52.5% respectively

Peak I and II: tryptophan-like substancesPeak III: the visible fulvic acid-like substance Peak IV: the UV fulvic acid-like substance

EffluentEffluent

EffluentInffluent

Microbiology characteristics at different temperature

Biomass analysis and SEM observation 

Temperature()  13 19 25 30

Biomass (mg/g) 6.9 9.4 11 14.1

13

19

2530

Bacilliform, filiform and spheroidal formTemperature had little influence on the carriers surface morphology, which consisted with the biomass analysis.

Microbiology characteristics at different temperature

Temperature( )  13 19 25 30

narG gene  (copies/g‐SS) 2.04× 109 1.59 ×109 1.65×109 7.19× 108

nirS gene (copies/g‐SS) 1.76× 109 1.95 × 109 1.20× 109 8.43× 108

nosZ gene (copies/g‐SS) 1.63 × 107 4.45× 106 2.56× 106 2.38× 106

The content of narG, nirS and nosZ genes at 13 and 19 conditions were higher than those under 25 and 30 . The highest abundance of narG, nirS and nosZ genes, i.e. 2.04×109 , 1.95×109 and 1.63×107copies/g-SS, were achieved at 13 , 19 and 13 respectively, which were consistent with the long 12h HRT compensation.

CONCLUSIONS5

CONCLUSIONS

1. The NO3--N removal and denitrification rate were not influenced by the

temperature of 13, 19, 25 and 30 at 12h HRT for polyethylene MBBR, the NO3

--N removal rate were 82.9±26.7 %, 80.1±27.2 %, 85.0±22.3 % and 82.9±13.3%. The long HRT of 12h may compensate for the denitrification ability of low temperature, which resulted the similar nitrate removal and denitrification rate at different temperatures.

2. The effluent COD could meet the Class IV limitation of Environmental Quality Standards for Surface Water (GB 3838-2002). Three-dimensional fluorescence spectra showed that both of the MBBR influent and effluent contained DOM, such as fulvic acid-like and tryptophan-like substance, etc., and the total fluorescence intensity removal were 47.6%, 49.0%, 50.5% and 52.5%, respectively, at different temperatures.

3. The abundance of narG, nirS and nosZ gene at 13 and 19conditions were higher than those under 25 and 30 . The highest abundance of narG and nosZ genes, i.e. 2.04×109 and 1.63×107copies/g-SS were achieved at 13 , which were consistent with the long 12h HRT compensation. The abundance of narG nitrate-reducing gene and nirS nitrite-reducing gene were both greater than that of nosZ nitrous oxide reducing gene.

4. Considering the nitrogen, organic pollutants removal efficiency and denitrification genes abundance as a whole, 25 was the optimum temperature for nitrogen removal of WWTP effluent by denitrification MBBR.

Acknowledgements

National Major Science and Technology Program for Water Pollution Control and Treatment (2014ZX07216-001)

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