Accepted Manuscript Start-up of Simultaneous Partial Nitrification, Anammox and Denitrification (SNAD) Process in Sequencing Batch Biofilm Reactor Using Novel Biomass Carriers Achlesh Daverey, Yi-Chian Chen, Kasturi Dutta, Yu-Tzu Huang, Jih-Gaw Lin PII: DOI: Reference:

S0960-8524(15)00243-6 http://dx.doi.org/10.1016/j.biortech.2015.02.064 BITE 14637

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

30 December 2014 12 February 2015 13 February 2015

Please cite this article as: Daverey, A., Chen, Y-C., Dutta, K., Huang, Y-T., Lin, J-G., Start-up of Simultaneous Partial Nitrification, Anammox and Denitrification (SNAD) Process in Sequencing Batch Biofilm Reactor Using Novel Biomass Carriers, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.02.064

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Start-up of Simultaneous Partial Nitrification, Anammox and Denitrification (SNAD) Process in Sequencing Batch Biofilm Reactor Using Novel Biomass Carriers Achlesh Daverey1, Yi-Chian Chen1, Kasturi Dutta1, Yu-Tzu Huang2, Jih-Gaw Lin1*

1

Institute of Environmental Engineering, National Chiao Tung University, Hsinchu, Taiwan –

30010 2

Department of Bioenvironmental Engineering, Chung-Yuan Christian University, 200 Chung

Pei Road, Chung-Li, Taiwan - 32023

*

Corresponding author

Jih-Gaw Lin, Institute of Environmental Engineering, National Chiao Tung University, Hsinchu, Taiwan – 30010, E-mail: [email protected] Tel.: +886 35722681 Fax: +886 35725958

1

Abstract Simultaneous partial nitrification, anammox and denitrification (SNAD) process was started-up in a 2.5 L sequencing batch biofilm reactor (SBBR) using novel biomass carriers. The SNAD process took only 51 d for start-up at nitrogen loading rate (NLR) and organic loading rate (OLR) of 120 and 60 g/m3-d, respectively. Long-term stable operation of SNAD process was observed at NLR and OLR of 360 and 180 g/m3-d with average total nitrogen and COD removal efficiencies of >88 and >90%, respectively. The values of conversion ratio Y( NO- + NO- ) / NH + 2

3

4

remained below 0.11 after the start-up period, which further confirmed the long-term stability of SNAD process. Results of polymerase chain reaction (PCR), qualitative PCR, and scanning electron microscopic (SEM) analysis of sludge samples confirmed the co-existence and enrichment of AOB, anammox bacteria and denitrifying bacteria in the reactor and biofilm formation on to the carriers.

Keywords: Anammox; SNAD; Start-up; Biofilm; Carrier

2

1. Introduction Anaerobic ammonium oxidation (Anammox) process, which directly converts ammonium and nitrite nitrogen to nitrogen gas, has been confirmed in the 1990s (Mulder et al., 1995). This novel process has been recognized as one of the most cost effective biological nitrogen removal processes. However, for practical application of anammox process in real wastewater treatment, it needs to be combined with partial nitrification (either in two reactor system or in a single reactor system), which provides nitrite for anammox bacteria. A combination of partial nitrification and anammox reactions in a single reactor i.e. Completely Autotrophic Nitrogen removal Over Nitrite (CANON) has been developed in 2001 (Third et al. 2001). Recently, integration of partial nitrification, anammox and denitrification reactions in a single reactor for treating wastewater having low carbon to nitrogen ratio (C/N) has been reported and the process named as Simultaneous partial Nitrification, Anammox and Denitrification (SNAD) (Chen et al., 2009). Anammox process when combined with partial nitrification (CANON or SNAD process) can save 63% aeration and 100% organic carbon source costs (Chen et al., 2012). The biomass yield of anammox bacteria is very low (0.11 g VSS/g NH4+-N, VSS - volatile suspended solids), which also saves sludge treatment costs (Jetten et al., 1998). The low biomass yield of anammox bacteria suggests that it have extremely low growth rates. The doubling time of anammox bacteria has been reported to be between 10-11 d with maximum specific growth rate of 0.0027 1/h (Strous et al., 1998; van der Star et al., 2007). A disadvantage of this low growth rate is the long start-up period required for anammox process, which limits its widespread application. Therefore, retention of anammox bacteria inside the reactor to minimize its washout and for stable operation is one of the main challenges for environmental engineers to reduce the duration of start-up periods.

3

Various reactor systems such as fluidized bed reactor (FBR, Mulder et al., 2005), rotating biological contactor (RBC, Egli et al., 2001), membrane bioreactor (MBR, van der Star et al., 2008), up-flow anaerobic sludge blanket (UASB, Schmidt et al. 2004.), gas lift reactor (Sliekers et al. 2003), sequencing batch reactor (SBR, Strous et al., 1998) have been used for the cultivation of anammox bacteria. Among these, SBR is considered to be the most suitable reactor for the growth of anammox bacteria due to efficient biomass retention, reliable operation for more than 1 year and simple set-up (Strous et al., 1998). Immobilization of anammox bacteria as biofilm on the surface of carrier can reduce the risk of biomass wash-out (Magri et al., 2012). Porous non-woven fabric (Chen et al., 2009), zeolite (Fernandez et al., 2008), novel acrylic resin material (Qiao et al., 2009), polyethylene sponge strips (Zhang et al., 2010), spherical plastic (Chen et al., 2012), bamboo charcoal (Chen et al., 2012) and polyurethane spheres (Daverey et al., 2013) have been used as carriers for immobilization of anammox bacteria. However, most of these carriers were used only to immobilize anammox bacteria, while in CANON and SNAD system other microbial species (AOB- ammonia oxidizing bacteria and denitrifiers) are also present. In this study, the SNAD process was started-up in a sequencing batch biofilm reactor (SBBR). Novel carriers were arranged inside the reactor for the biomass retention and their effectiveness in reducing the start-up time of SNAD process in SBBR was evaluated.

2. Materials and methods 2.1 Biomass carrier The biomass carriers (Fig. S1) used in this study were prepared by using waste activated sludge (WAS) collected from the municipal wastewater treatment plant located in Taiwan, red soil and

4

chemical additive vesicant. The red soil was added to provide stickiness while chemical additivevesicant provided hardness to the carrier. Details of the carrier formation and characteristics can be found elsewhere (Daverey et al., 2014; Su 2008). In brief, the components of the carrier were dried (between 80oC and 120oC), crushed and sieved to obtain coarse powder (particles diameters of about 0.3 mm). The coarse powder was further grinded to obtain fine powder (particles diameters of ≤10 µm). The components were mixed with final composition ratio (w/w) of WAS, red soil and chemical additive vesicant as 5:6:1 to 5:12:1 in the carrier, and molded at 1000oC firing followed by cooling to solidify the carriers. Total forty carriers were used and arranged around the wall of the reactor (Fig. S1). The diameter, surface area, uniaxial compressive strength, bulk density and water absorption capacity of the carriers used were 2 cm, 12.6 cm2/carrier, 36.7 Kgf/cm2, 7.1 g/cm3 and 32.99%, respectively (Su 2008).

2.2 Sequencing batch biofilm reactor (SBBR) and its operation The SBBR used in this study to develop SNAD process had a working volume of 2.5 L. The reactor was equipped with pH, oxidation reduction potential (ORP; Suntex PC3200, Taiwan) and dissolved oxygen (DO; HACH sc100, Germany) probes for online monitoring of pH, ORP and DO, respectively. The reactor was operated at a fixed speed of 150 rpm by an overhead mechanical stirrer. Two separate pumps were used to supply influent to the reactor and withdraw effluent from the reactor. The SBBR was operated at a fixed temperature of 25oC. The pH of the reactor content was controlled between 7 and 8 by adding NaHCO3 as buffering agent. The DO concentration in the reactor was maintained at ~0.1 mg O2/L. The SBBR was operated in cycles of 24 h. The distribution of each cycle consists of 23.75 h for reaction (including 6 h for filling), 0.1 h for settling and 0.25 h for decanting. The feeding time

5

(6 h) was divided into 10 minutes of fast feeding (~0.8 L) and 5 h and 50 minutes for slow feeding (~0.8 L). Fast feeding was done to avoid long time air exposure of carriers. Slow feeding was done to avoid the shock loading. The reactor was operated in four different stages and in these stages nitrogen loading rate (NLR) and organic loading rate (OLR) were stepwise increased from 80 to 360 g/m3-d and 40 to 180 g/m3-d, respectively (Table 1).

2.3 Inoculum sludge The SBBR was inoculated with 1.5 L of sludge collected from a full-scale landfill leachate treatment plant located in Xin-Feng, Taiwan. The initial biomass concentration and the specific anammox activity were 4.5 g VSS/L and 0.05 g N2 /g VSS-d, respectively. The polymerase chain reaction (PCR) experiments were performed, which confirmed the presence of AOB and anammox bacteria. No clear bands were observed for denitrifying bacteria and NOB.

2.4 Influent wastewater composition Synthetic wastewater prepared in the laboratory was used as influent in this study. Ammonium nitrogen (NH4+-N) and organic matter (COD) were supplemented in the form of NH4Cl (ammonium chloride) and glucose, respectively. The influent NH4+-N to COD ratio was fixed at 0.5. The synthetic wastewater was supplemented with mineral medium. The composition of the mineral medium was (g/L except for trace element solution): KHCO3, 1.25; KH2PO4, 0.025; CaCl2.2H2O, 0.3; MgSO4, 0.2; FeSO4, 0.00625; EDTA, 0.00625 and trace element solution 1 mL/L (Sliekers et al., 2002). The stock solutions of mineral medium and trace elements were stored in a refrigerator at 4oC.

6

2.5 Scanning electron microscope (SEM), PCR and qPCR analysis Two carriers were taken out from the reactor at the end of experiment for SEM analysis. Carriers were dipped in 100 ml reactor effluent (or buffer solution) and sonicated for 10 min to remove attached biomass from the carrier. The removed biomass from the carrier was used for SEM analysis according to the methods reported by de Graaff et al. (2011). In brief, sludge samples were washed with phosphate buffer (0.1, M; pH, 7.0) and were fixed with 2.5% glutaraldehyde solution at 4°C for overnight. After fixation, samples were washed twice in phosphate buffer and subsequently, dehydrated with ethanol solutions graded from 50-100% for 20 min each. Samples were dried by critical point drying (liquid CO2) before SEM (Hitachi S-4700I, Japan) observations. The PCR and qPCR experiments were performed as reported earlier (Daverey et al., 2013) using the suspended biomass withdrawn from the SBBR. In brief, the total genomic DNA from the suspended biomass was extracted and the DNA concentration was determined on a photometer Gene Quant pro (Amersham Biosciences, Pittsburg, PA, USA). Table 3 shows the primers used for identification of microbial species (AOB, NOB, denitrifying bacteria, anammox bacteria, Candidatus Kuenenia stuttgartiensis (KS) Candidatus Brocadia anammoxidans (BA), eubacteria and most of the anammox) by PCR and qPCR analysis. A 96 well Gradient Palm-Cycler (Corbett Research Pty Ltd., Austria) was used for PCR amplification. A mixture of DNA template (1 µl; about 50 ng), each primer (1 µl; 10 µM) as described in Table 3, sterile water (9.5 µl) and 2×Taq PCR Master Mix (Genomics BioSd & Tech, Taiwan; 12.5 µl) was used in each PCR reaction. For qPCR experiments, the DNA (1 µl; about 5 ng), each primer (0.5 µl; 10 µM) as described in Table 3, sterilized water (3 µl) and fluorescent dye SsoFastTM EvaGreen® Supermix (BIO-RAD, USA; 5 µl) were mixed for each reaction. The DNA was amplified in a 96 well Gradient Palm-

7

Cycler (Corbett Research Pty Ltd., Austria) with a following thermal profile: initial denaturation at 95°C for 30s, 1 cycle; denaturation at 95°C for 5s, 40 cycles; annealing at 54°C (BACT1369F/PROK1492R) or 58°C (Amx809F/Amx1066R) for 5s followed by a dissociation step (95°C for 15 seconds, 65°C for 15 seconds, and a slow ramp at 95°C). The specificities of the PCR and qPCR were examined by Agarose gel (1%) electrophoreses as reported earlier (Han et al., 2013).

2.6 Analytical methods The samples were collected twice or thrice per week from the reactor for analysis. The measurements of suspended solid (SS), volatile suspended solid (VSS), alkalinity, NH4+-N, NO2-N, NO3--N and COD were analyzed according to the Standard methods (APHA, 1998).

3. Results and discussion 3.1 Fast start-up of SNAD process (Stage I and II) The reactor was started with NLR, OLR and HRT of 80 g/m3-d, 40 g/m3-d and 2.5 d, respectively. The DO concentration during the reaction period of SBR cycle was intended to maintain at ~0.1 mg O2/L by adjusting the air flow-rate during each stage (Fig. 1 a). The DO supply automatically turned off as the value increased above 0.5 mg/L. Besides, nitrite was analyzed every day to avoid nitrite accumulation in the reactor during the start-up period (1-51 d). This low DO (88% TN removal and >90% COD removal) of SNAD process in SBBR was also observed. SEM analysis of sludge samples immobilized on to the carriers confirmed the biofilm formation on to the carriers. The results of PCR and qPCR further confirmed the co-existence and enrichment of microbial communities of SNAD process i.e. AOB, anammox bacteria and denitrifying bacteria in the SBBR.

Acknowledgement

We thank Leaderman & Associates Co., Ltd., Taiwan for partially funding this research work.

References

1. APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC. 2. Braker, G., Fesefeldt, A., Witzel, K.P., 1998. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Applied and Environmental Microbiology ,64 (10), 3769-3775. 3. Braker, G., Tiedje, J.M., 2003. Nitric Oxide Reductase (norB) genes from pure cultures and environmental samples. Applied and Environmental Microbiology, 69, 3476–3483. 14

4. Chen, H.H., Liu, S.T., Yang, F.L., Yuan, X., Wang, T., 2009. The development of simultaneous partial nitrification, Anammox and denitrification (SNAD) process in a single reactor for nitrogen removal. Bioresource Technology, 100, 1548–1554. 5. Chen, C. j., Huang, X.X., Lei, C.X., Zhu, W.J., Chen, Y.X., Wu, W.X., 2012. Improving Anammox start-up with bamboo charcoal. Chemosphere, 89, 1224-1229. 6. Daverey, A., Su, S.H., Huang, Y.T., Lin, J.G., 2012. Nitrogen removal from optoelectronic wastewater using the simultaneous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in sequencing batch reactor. Bioresource Technology, 113, 225-231. 7. Daverey, A., Su, S.H., Huang, Y.T., Chen, S.S., Sung, S., Lin, J.G., 2013. Partial nitrification and anammox process: a method for high strength optoelectronic industrial wastewater treatment. Water Research, 47(9), 2929-2937. 8. Daverey, A., Chen, Y.C., Sung, S., Lin, J.G., 2014. Effect of zinc on anammox activity and performance of simultaneous partial nitrification, anammox and denitrification (SNAD) process. Bioresource Technology, 165, 105-110. 9. de Graaff, M.S., Temmink, H., Zeeman, G., van Loosdrecht, M.C., Buisman, C.J., 2011. Autotrophic nitrogen removal from black water: calcium addition as a requirement for settleability. Water Research, 4 5, 63-74. 10. Egli, K., Fanger, U., Alvarez, P.J., Siegrist, H., van der Meer, J.R., Zehnder, A.J., 2001. Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Archives of Microbiology, 175, 198-207.

15

11. Fernandez, I., Vazquez-Padin, J.R., Mosquera-Corral, A., Campos, J.L., Mendez, R., 2008. Biofilm and granular systems to improve Anammox biomass retention. Biochemical Engineering Journal, 42, 308-313. 12. Han, P., Huang, Y.T., Lin, J.G., Gu, J.D., 2013. Comparison of two 16S rRNA genebased PCR primer sets in unraveling Anammox bacteria from different environmental samples. Applied Microbiology and Biotechnology, 97, 10521-10529. 13. Jetten, M.S., Strous, M., van De Pas-Schoonen, K.T., Schalk, J., van Dongen, U.G., van De Graaf, A.A., Logemann, S., Muyzer, G., Van Loosdrecht, M.C., Kuenen, J., 1998. The anaerobic oxidation of ammonium. FEMS Microbiology Reviews, 22 (5), 421–437. 14. Lan, C-J., Kumar, M., Wang, C-C., Lin, J-G., 2011. Development of simultaneous partial nitrification, anammox and denitrification (SNAD) process in a sequential batch reactor. Bioresource Technology, 102, 5514-5519. 15. Li, M., Ford, T., Li, X., Gu, J.D., 2011. Cytochrome cd1-containing nitrite reductase encoding gene nirS as a new functional biomarker for detection of anaerobic ammonium oxidizing (Anammox) bacteria. Environmental Science and Technology, 45 (8), 35473553. 16. Magri, A., Vanotti, M.B., Szogi, A.A., 2012. Anammox sludge immobilized in polyvinyl alcohol (PVA) cryogel carriers. Bioresource Technology, 114, 231-240. 17. Mulder, A., van de Graaf, A.A., Robertson, L.A., Kuenen, J.G., 1995. Anaerobic ammonium oxidation discovered in a denitrifying fluidized-bed reactor. FEMS Microbiology Ecology, 16 ( 3), 177-178.

16

18. Mulder, A., Vandegraaf, A.A., Robertson, L.A., Kuenen, J.G., 1995. Anaerobic ammonium oxidation discovered in a denitrifying fluidized-bed reactor. FEMS Microbiology Ecology, 16 (3), 177-183. 19. Penton, C.R., Devol, A.H., Tiedje, J.M., 2006. Molecular evidence for the broad distribution of anaerobic ammonium-oxidizing bacteria in freshwater and marine sediments. Applied and Environmental Microbiology, 72, 6829-6832. 20. Qiao, S., Kawakubo, Y., Cheng, Y., Nishiyama, T., Fujii, T., Furukawa, K., 2009. Identification of bacteria coexisting with anammox bacteria in an upflow column type reactor. Biodegradation 20, 117–124. 21. Rotthauwe, J.H., Witzel, K.P., Liesack, W., 1997. The ammonia monooxygenase structural gene amoA as a functional maker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology, 63, 47044712. 22. Schmidt, J.E., Batstone, D.J., Angelidaki, I., 2004. Improved nitrogen removal in upflow anaerobic sludge blanket (UASB) reactors by incorporation of Anammox bacteria into the granular sludge. Water Science and Technology, 49 (11-12), 69-76. 23. Schmid, M., Walsh, K., Webb, R., Rijpstra, W.I., van de Pas-Schoonen, K., Verbruggen, M.J., Hill, T., Moffett, B., Fuerst, J., Schouten, S., Sinninghe Damsté, J.S., Harris, J., Shaw, P., Jetten, M., Strous, M., 2003. Candidatus “Scalindua brodae”, sp. nov., Candidatus “Scalindua wagneri”, sp. nov., two new species of anaerobic ammonium oxidizing bacteria. Systematic and Applied Microbiology, 26, 529-538.

17

24. Sliekers, A.O., Derwort, N., Campos Gomez, J.L., Strous, M., Kuenen, J.G., M.S.M, Jetten., 2002. Completely autotrophic ammonia removal over nitrite in one reactor. Water Research, 36, 2475-2482. 25. Su, W.F. 2008. Application of recycle porous diffusers in a SND-sequencing batch biofilm reactor (SBBR). Thesis of the Institute of Environmental Science and Engineering (Tunghai University). 26. Suzuki, M.T., Taylor, L.T., DeLong, E.F., 2000. Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5'-nuclease assays. Applied and Environmental Microbiology, 66 (11), 4605-4614. 27. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M., 1998. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammoniumoxidizing microorganisms. Applied Microbiology and Biotechnology, 50, 589-596. 28. Third, K.A., Sliekers, A.O., Kuenen, J.G., Jetten, M.S.M., 2001. The CANON system (completely autotrophic nitrogen-removal over nitrite) under ammonium limitation: Interaction and competition between three groups of bacteria. Systematic and Applied Microbiology, 24, 588-596. 29. Tsushima, I., Kindaichi, T., Okabe, S., 2007. Quantification of anaerobic ammoniumoxidizing bacteria in enrichment cultures by real-time PCR. Water Research, 41 (4), 785794. 30. Trigo, C., Campos, J.L., Garrido, J.M., Mendez, R., 2006. Start-up of the anammox process in a membrane bioreactor. Journal of Biotechnology, 126 (4), 475-487. 31. Van der Star, W.R.L., Abma, W.R., Blommers, D., Mulder, J.W., Tokutomi, T., Strous, M., Picioreanu, C., Van Loosdrecht, M.C.M., 2007. Startup of reactors for anoxic

18

ammonium oxidation: Experiences from the first full-scale anammox reactor in Rotterdam. Water Research, 41, 4149-4163. 32. Van der Star, W.R., Miclea, A.I., van Dongen, U.G., Muyzer, G., Picioreanu, C., van Loosdrecht, M.C., 2008. The membrane bioreactor: a novel tool to grow anammox bacteria as free cells. Biotechnology and Bioengineering, 101, 286-294. 33. Vazquez-Padin, J.R., Pozo, M.J., Jarpa, M., Figueroa, M., Franco, A., Mosquera-Corral, A., Campos, J.L., Méndez, R., 2009. Treatment of anaerobic sludge digester effluents by the CANON process in an air pulsing SBR. Journal of Hazardous Materials, 166 (1), 336341. 34. Wang, T., Zhang, H.M., Yang, F.L., Liu, S.T., Fu, Z.M., Chen, H.H., 2009. Start-up of the Anammox process from the conventional activated sludge in a membrane bioreactor. Bioresource Technology, 100, 2501-2506. 35. Zhang, Z., Chen, S., Wu, P., Lin, L., Luo, H., 2010. Start-up of the Canon process from activated sludge under salt stress in a sequencing batch biofilm reactor (SBBR). Bioresource Technology, 0101, 6309-6314.

19

Figure captions Fig. 1 Profiles of (a) DO and oxygen supply, and (b) pH and alkalinity during stages I-IV in the

SBBR. Fig. 2 Temporal variations of (a) the nitrogen compounds in influent and effluent along with

nitrogen removal efficiencies, and (b) COD in influent and effluent along with COD removal efficiency in SBBR. Fig. 3 Temporal variations of biomass and solids concentration during stages I-IV in the reactor. Fig. 4 Results of PCR by performing agarose gel electrophoreses of sludge samples taken on 235

d.

20

Table 1 Operating conditions in SBBR. Parameter

Stages I

II

III

IV

1-16

17-75

76-134

135-248

1

1.5

1.5

1.5

HRT (d)

2.5

1.67

1.67

1.67

COD (mg/L)

100

100

200

300

NH4+-N (mg/L)

200

200

400

600

Organic loading rate (g/m3.d)

40

60

120

180

Nitrogen loading rate (g/m3.d)

80

120

240

360

Duration (d) Flow (L/d)

21

Table 2 Specific primers used for identification of microbial species in PCR and qPCR analysis. Microorganisms/Strain

Specific primer

Reference

Ammonia oxidizing bacteria (AOB)

amoA-1F/amoA-2R

Rotthauwe et al., 1997

Nitrite oxidizing bacteria (NOB)

nirS-1F/nirS-6R

Braker et al., 1998

Denitrifying bacteria

cnorB-2F/cnorB-6R

Braker et al., 2003

Anammox bacteria

Brod541F/Amx820R

Penton et al., 2006

Anammox bacteria

AnnirS379F/AnnirS821R Li et al., 2011

Candidatus Kuenenia stuttgartiensis (KS)

KS-qF3/KS-qR3

Daverey et el., 2013

Candidatus Brocadia anammoxidans (BA)

BAqF/BAqR

Daverey et el., 2013

Eubacteria

BACT1369F/PROK1492R

Suzuki et al., 2000

Most of the Anammox

Amx809F/Amx1066R

Tsushima et al., 2007

22

Table 3 Comparison of start-up of Anammox-related processes using different carriers. b

Carrier

Reactor a System

Process

Seed Sludge

Volume (L)

Start-up time (d)

Influent (mg-N/L)

NLR 3 (g/m -d)

HRT (d)

Reference

Membrane

MSBR

Anammox

A

5

350

400

400

1

Zeolite

SBR

Anammox

B

5

160

600

600

1

Membrane

MBR

Anammox

C

4.8

59

100

50

2

Non-woven

SBBR

CANON

D

6.5

86

200

100

2

Polyurethane

SBR

CANON

E

18

164

200

33

6

Non-woven

NRBC

SNAD

F

-

46/181

200

66

3

WAS pellet

SBR

SNAD

G

2.5

51

200

120

1.67

Trigo et al. (2006) Fernandez et al. (2008) Wang et al. (2009) Zhang et al. (2010) Daverey et al. (2013) Chen et al. (2009) This study

c

a

MSBR = Membrane Sequencing Batch Reactor; NRBC = Non-woven Rotating Biological Contactor A = Enriched Anammox granular sludge; B = Enriched Anammox sludge; C = Aerobic and nitrifying activated sludge; D = Activated sludge (Anammox and aerobic nitrifiers); E = Enriched SNAD seed sludge; F = Enriched Anammox and partial nitrification biomass; G = SNAD seed sludge c CANON to SNAD within 46 days; Anammox to SNAD within 181 days b

23

Fig. 1

24

Fig. 2

25

Fig. 3

26

Fig. 4

27

Highlights

•

Novel carriers used were highly efficient in retaining biomass inside the reactor.

•

SNAD reactor was started-up in a short period (51 d).

•

Long-term stable operation of SNAD process in SBBR was observed.

•

SEM analysis confirmed biofilm formation on to the carriers.

•

qPCR confirmed the enrichment of AOB, anammox bacteria and denitrifying bacteria.

28