Tolerance Level of Dissolved Oxygen to Feed into Anaerobic Ammonium Oxidation (anammox) Reactor

ABSTRACT In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study effects of influent dissolved oxygen (DO) on anammox activity since effluent from nitritation process feed to anammox process. In this study, the effects of influent DO on anammox bacteria entrapped in gel carriers were investigated using continuous feeding tests. The tests were performed in duplicate to confirm the reproducibility and anammox activities were evaluated under different DO concentration of 0 from 5 mg/L in influent. These results suggested that the DO concentration in influent to anammox reactor must be less than 2.5 mg/L. In addition, it was shown that the effect of influent DO on the anammox reaction is reversible because fallen anammox activity by influent DO of 5 mg/L recovered when the influent DO concentration was decreased to less than 1 mg/L.

Tolerance Level of Dissolved Oxygen to Feed into Anaerobic Ammonium Oxidation (anammox) Reactor Yuya KIMURA*, Kazuichi ISAKA*, Futaba KAZAMA**

*Hitachi Plant Technologies, Ltd., Chiba 271-0064, Japan

**Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi 400-8511, Japan

ABSTRACT

In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study effects of influent dissolved oxygen (DO) on anammox activity since effluent from nitritation process feed to anammox process In this study, the effects of influent DO on anammox bacteria entrapped in gel carriers were investigated using continuous feeding tests The tests were performed in duplicate to confirm the reproducibility and anammox activities were evaluated under different DO concentration of 0 from 5 mg/L in influent These results suggested that the DO concentration in influent to anammox reactor must

be less than 2.5 mg/L In addition, it was shown that the effect of influent DO on the anammox reaction is reversible because fallen anammox activity by influent DO of 5 mg/L recovered when the influent DO concentration was decreased to less than 1 mg/L

Keywords: anammox, dissolved oxygen, immobilization

INTRODUCTION

Several kinds of wastewater, for example, wastewater from the dewatering of digested sludge and landfill leachate, have high concentrations of ammonium and low concentrations of biodegradable organic compounds (low C/N ratio) In general, nitrogen is removed by using a nitrification-denitrification process However, in order to achieve complete biological nitrogen removal during the denitrification of these kinds

of wastewater, an additional organic carbon source must be added, resulting in higher operating costs Therefore, the conventional nitrification-denitrification process is not ideal for ammonium-containing wastewater with low C/N ratios

In the 1990s, a novel metabolic pathway, anaerobic ammonium oxidation (anammox),

was discovered (Mulder et al., 1995; Van de Graaf et al., 1996) In this process, since

ammonium is used with nitrite as the electron donor, addition of organic compounds is not required because anammox bacteria are autotrophic We are developing a novel nitrogen removal system using anammox bacteria immobilized in a gel carrier Anammox bacteria immobilized in a gel carrier are easily separated from the liquid in the reactor, and this yields prolonged biomass retention times even with short hydraulic

retention times (HRT) (Isaka et al., 2007) Therefore, stable and high nitrogen removal

performance can be attained by anammox gel carriers

Since nitrite is used in anammox reaction, a pre-treatment process to oxidize ammonium

to nitrite (nitritation) is needed In most of the treatment systems using anammox, a two

step system of nitritation-anammox is adopted (Hellinga et al., 1998; Van der Star et al., 2007; Yamamoto et al., 2008) We are developing a two step system of

nitritation-anammox in a similar way As a matter of course, the nitritation process is

carried out in an aerobic condition The DO range in our nitritation process is set to 2

mg/L basically, and it is controlled from 1 to 4 mg/L (Isaka et al., 2009) Tokutomi

(2004) operated a nitritation process having aerobic granular biomass at DO concentration of less than 1 mg/L

On the other hand, the anammox process is in an anaerobic condition Consequently, effluent from the nitritation process is fed into the subsequent anammox process as influent with dissolved oxygen (DO) There is a possibility that the anammox activity may be affected by this However, there are few reports in the literature about the effects

of influent DO on anammox activity although a two step system of nitritation-anammox

is adopted in many cases

It is necessary to investigate DO range of influent in order to operate a stable anammox process The purpose of this investigation was to better understand the influence of influent DO, with assumed nitritation on anammox activity in the gel carrier We also investigated ways to overcome this influence

MATERIALS AND METHODS

Seed anammox sludge

Enriched sludge was grown in a 22 L fixed bed reactor filled with porous polyester

non-woven fabric carriers (Furukawa et al., 2002) at 36°C Synthetic medium

(described below) was continuously fed into the reactor In order to collect the enriched sludge adhering to the non-woven fabric carriers, the non-woven fabric was washed in a tank filled with the effluent water from the enrichment reactor The sludge that settled in the tank was collected and used for gel entrapment

Gel carrier

Anammox sludge was entrapped in a polyethylene glycol (PEG) gel carrier (Sumino et

al., 1997; Isaka et al 2007) First, a PEG prepolymer and a promoter, (N, N, N’, N’ -

tetramethylenediamine), were dissolved in water Then, the resulting mixture and anammox bacteria enriched sludge were mixed in a beaker To initiate polymerization,

an initiator (potassium persulfate) was added to the beaker The polymerized carrier gel was cut into 3 mm cubes The gel carrier contained 15% (w/v) PEG, 0.5% (w/v) promoter, 0.25% (w/v) initiator, and 0.4% (w/v) anammox-bacteria enriched sludge

Synthetic medium

For continuous feeding tests, a synthetic medium was used The medium contained: 750 mg/L (NH4)2SO4; 1000 mg/L NaNO2; 420 mg/L NaHCO3; 27.2 mg/L KH2PO4; 300 mg/L MgSO4·7H2O; 180 mg/L CaCl2·2H2O; and 1 mL of trace element solutions 1 and

2 Trace element solution 1 contained 5 g/L EDTA and 5 g/L FeSO4·7H2O Trace element solution 2 contained 15 g/L EDTA; 0.43 g/L ZnSO4·7H2O; 0.24 g/L CoCl2·6H2O; 0.99 g/L MnCl2·4H2O; 0.25 g/L CuSO4·5H2O; 0.22 g/L Na2MoO4·2H2O; 0.19 g/L NiCl2·6H2O; 0.21 g/L NaSeO4·10H2O; and 0.014 g/L H3BO4

Chemical analysis and calculations

Both the influent and the effluent of the anammox reactors were analyzed in order to evaluate the anammox performances The ammonium concentrations were measured by

using the indophenol method (Weatherburn, 1967) The nitrite and nitrate concentrations were determined by using ion chromatography (ICS-1500, Dionex, USA)

The nitrogen loading rate and conversion rate was calculated from the influent and

effluent ammonium and nitrite concentrations as follow

Nitrogen Loading Rate (kg-N/m3/d)

= (inf.NH4-N(mg/L) + inf.NO2-N(mg/L)) × ×10-3 Nitrogen Conversion Rate (kg-N/m3/d)

= {(inf.NH4-N– eff.NH4-N) (mg/L) + (inf.NO2-N – eff.NO2-N) (mg/L) } × ×10-3

Reactor and experimental setup

A cylindrical reactor containing the anammox bacteria entrapped in the gel carrier was

used for continuous water treatment test (Fig 1) The volume of the reactor was 500

mL, and 100 mL of the gel carrier cubes was placed inside the reactor (packing ratio, 20%) The reactors were operated in a temperature-controlled room at 30°C The gel carriers were agitated continuously Agitation was necessary primarily to mix the influent and to remove the nitrogen gas bubbles that formed on the surfaces of the gel carriers HRT was set to 2 hours The to in the reactor was monitored and adjusted pH 7.6 by adding 0.2 N hydrochloric acid We operated two reactors that nitrogen conversion rates were about 2.9 kg-N/m3/d (reactor 1) and 4.3 kg-N/m3/d (reactor 2) in order to evaluate the reproducibility of the test The difference of nitrogen conversion rates between reactor 1 and 2 depends on cultivation term and the amount of bacteria in

each reactor (Isaka et al., 2007)

Since the DO range in our nitritation-process is from 1 to 4 mg/L (Isaka et al., 2009),

maximum DO concentration in this study was set to 5 mg/L that allow some latitude

24 HRT

24 HRT

DO sensor

DO Controller

N 2 gas

Synthetic medium

Stirrer

Separator

Effluent

Influent

pH sensor 0.2N HCl

Gel Carrier Influent pump

Stirrer

P

P

Effluent Influent

Reactor 2

Reactor 1

Fig 1 - Schematic illustration of the reactors used in the continuous feeding tests using

anammox bacteria entrapped in gel carriers

DO concentrations of the synthetic medium in an influent tank were adjusted from 0 to

5 mg/L by using DO controller and sparging with nitrogen gas and air blower before feeding the synthetic medium to each reactor

RESULTS AND DISCUSSION

Effect of influent DO concentration on anammox activity

The effects of influent DO on anammox activities in the gel carrier were evaluated using two continuous feeding tests with each reactor Fig 2 shows the nitrogen loading rate and the conversion rates over time of reactor 1 and 2 The influent DO concentration was increased to about 5.0 mg/L from 0.2 mg/L stepwise The influent DO concentration was increased to next step, when the nitrogen conversion rate had not changed greatly

(a)

(b)

0.0 1.0 2.0 3.0 4.0 5.0

0.0 1.0 2.0 3.0 4.0 5.0

3 /d)

Time (day)

0.0 1.0 2.0 3.0 4.0 5.0

0.0 1.0 2.0 3.0 4.0 5.0

3 /d)

Time (day)

Fig 2 - The nitrogen loading rate and the conversion rates over time

(a) Reactor 1 (b) Reactor 2 Nitrogen loading rate (closed triangles), nitrogen conversion rate of reactor 1 (open circles), nitrogen conversion rate of reactor 2 (open squares) and the solid line (no symbol) shows influent DO concentration

In reactor 1, a nitrogen conversion rate of about 2.9 kg-N/m3/d was maintained with influent ammonium and nitrite concentrations of about 160 and 190 mg-N/L, respectively When DO of 2 mg/L was fed into the reactor, the nitrogen conversion rate

of about 2.9 kg-N/m3/d was maintained The DO of influent was set up over 3.0 mg/L, anammox activity gradually decreased The nitrogen conversion rate was 1.3 kg-N/m3/d when DO of 5 mg/L was fed into the reactor

In reactor 2, a nitrogen conversion rate of about 4.3 kg-N/m3/d was maintained The influent DO was set up over 3.0 mg/L, anammox activity gradually decreased The nitrogen conversion rate was 2.3 kg-N/m3/d when DO of 5 mg/L was fed into the reactor When DO of about 4 mg/L, 2.5 mg/L and less than 1 mg/L were fed into the reactors, DO concentrations in the effluent were confirmed about 3 mg/L, 2 mg/L and less than 1 mg/L, respectively

These results showed that an anammox activity is affected by influent DO because the anammox activities decreased when DO of high concentration was fed into the reactor

of each

Fig 3 shows the amounts of nitrite removed (NO2-Nre.) and nitrate produced (NO3-Npro.) based on the amount of ammonium removed (NH4-Nre.) in these reactors

in each influent DO concentration Strous et al (1998) have proposed that the anammox

reaction can be expressed as follows:

NH4+ + 1.32NO2– + 0.066HCO3– + 0.13H+

→ 1.02N2 + 0.26NO3– + 0.066CH2O0.5N0.15 + 2.03H2O

0.0

0.5

1.0

1.5

2.0

0.0 0.2 0.4 0.6 0.8

Influent DO concentration on average

Fig 3 - The amounts of nitrite removed (NO2-Nre.) and nitrate produced (NO3-Npro.)

based on the amount of ammonium removed (NH4-Nre.) of these reactors in each influent DO concentration NO2-Nre./NH4-Nre in reactor 1 (closed circles) and reactor 2 (closed squares) NO3-Npro./NH4-Nre in reactor 1 (open

circles) and reactor 2 (open squares)

In their case, therefore, NH4-Nre.:NO2-Nre.:NO3-Npro is 1:1.32:0.26 In our results, the average amounts for whole performed period were calculated to be 1:1.20 ± 0.18:0.22 ± 0.02 in reactor 1 and 1:1.25 ± 0.16:0.21 ± 0.03 in reactor 2 These results are very close

to the reported value (Strous et al., 1998) Moreover, these ratios did not show much

difference even when influent DO concentration was different When DO concentration

of 0.2 and 5 mg/L were fed into these reactors, the average ratios of both reactors were calculated to be 1:1.35:0.19 and 1:1.31:0.23, respectively

Therefore, it is suggested that the denitrification performances in our tests under high

DO concentration were also mainly anammox reaction and other denitrification and nitrification were comparatively minor in our tests

Relationship between anammox activity and influent DO concentration

Fig 4 shows the relationships between anammox activities and influent DO

concentrations of the two reactors The maximum nitrogen conversion rate of each reactor at the influent DO of about 0.2 mg/L was set to an anammox activity value of 1.0, and for the other concentrations of influent DO, the anammox activity was plotted

as a function of influent DO concentration The two reactors showed very similar behavior Neither anammox activity was affected when the influent DO of each was less than about 2.5 mg/L Influent DO of more than 3.0 mg/L affected anammox activity When influent DO in reactor was 5 mg/L, the anammox activity decreased by about 45% These results suggested that perhaps the boundary, where anammox activity is affected by influent DO, is around 2.5 mg/L

There are few effects of DO in effluent of a nitritation process on an anammox process,

in the case that operating DO in the nitritation process is around 2 mg/L In a real full-scale system, the DO tolerance level for anammox process may be higher because

DO is consumed by an aerobic bacteria including nitrifying bacteria in the effluent of nitritation process

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Influent DO concentration (mg/L)

Fig 4 - The Relationship between anammox activity ratio and influent DO

concentration Reactor 1 (open circles) and reactor 2 (open squares)

In this study, it is very likely that most of the DO in the influent tank is fed into a reactor, because HRT was very short (2 hours) DO concentration in the effluent was confirmed about 2 mg/L when DO of 2.5 mg/L was fed into the reactor 1 Moreover, there may be few cases in a real full-scale system that HRT is longer than 2 hours Therefore, our results were able to confirm the actual influence of influent DO on anammox activity Incidentally, the DO loading rate in a case that influent DO concentration was 2.5 mg/L in our test is 0.03 kg/m3/d

The influent DO concentrations were changed with different nitrogen conversion rates

in this study In these results, even when nitrogen conversion rates between each reactor were different (2.9 and 4.3 kg-N/m3/d), we confirmed that there is no significant difference in the influence of influent DO It is thought that the amount of anammox bacteria was different between reactor 1 and 2, because the nitrogen conversion rate was

different According to the result of real-time PCR (Isaka et al., 2007), the amount of

16S rRNA gene derived from the Planctomycales are calculated at 4.2 × 109 copies/g-carrier in reactor 1 and 1.8 × 1011 copies/g-carrier in reactor 2 Therefore, it is thought that this difference of amount does not show the effect of influent DO on anammox activity

These results were gotten in about one month Liu et al (2008) investigated anammox

consortium using wastewater containing DO for long term However, the results showed that the anammox activity was decreased only 5% because Nitrosomonas protected anammox bacteria Planctomycetales against oxygen Therefore, the effects may not be different, even if we operate the reactors for a longer term

Nowadays, a single step system that combines a nitritation process and anammox

process is being developed in aerobic condition (Third et al., 2001; Slinkers et al., 2002; Furukawa et al., 2006) In the system both nitrifying and anammox bacteria can be in

single reactor because oxygen zone and no oxygen zone are formed in the biofilm

Furukawa et al (2006) carried out SNAP (Single-stage Nitrogen removal Anammox

and Partial nitritation) process as a single step system under operational condition of

DO 2 - 3 mg/L

In the case of the single reactor, some oxygen may reach the zone that anammox bacteria exist, even though oxygen is consumed by nitrifying bacteria because the reactor is always aerobic condition of about 2 mg/L In this study, anammox activity was confirmed on condition that influent DO was 2.5 mg/L and the effluent DO was about 2 mg/L It is thought that anammox bacteria can maintain their activity even when some oxygen exists around the bacteria, though DO is not measured around anammox bacteria and a single step system cannot be compared with our system Therefore, it is suggested that we don’t need to be afraid of DO of at least 1 mg/L when anammox reactor has enough their activity

Recovery from fallen anammox activity

There is a possibility that influent DO concentration is increased due to control problems during the nitritation process and then anammox activity is decreased by this

DO

It was examined whether fallen anammox activity by influent DO can be recovered

when influent DO concentration is decreased Fig 5 shows influent DO concentration and nitrogen conversion rates over time of two reactors In reactor 1, the nitrogen conversion rate had decreased from 2.9 to 1.3 kg-N/m3/d when influent DO of 5 mg/L had been fed into reactor 1 After that, the DO concentration was decreased to less than 1.0 mg /L Then, the nitrogen conversion rate increased

In reactor 2, the nitrogen conversion rate, that was decreased to 2.3 from 4.3 kg-N/m3/d

by influent DO of 5 mg/L, increased when DO of less than 1.0 mg/L was fed into reactor 2 Anammox activities in both reactors recovered to about 80% within 3 days These results show that the anammox activity can be recovered by decreasing DO concentration of influent Therefore, the effect of influent DO on the anammox bacteria

is reversible We suggest that the DO in the anammox process should be decreased immediately using sparging N2 or Ar gas when the anammox process is inhibited by influent DO Effluent DO from nitritation process should be controlled

CONCLUSIONS

In this study, the effects of influent DO on anammox bacteria entrapped in gel carriers were investigated using continuous feeding tests for one month Anammox activity was affected by influent DO of over 2.5 mg/L The effect of influent DO on the anammox bacteria is reversible Therefore, there are few effects of DO in effluent from a nitritation process on an anammox process, in the case that operating DO in the nitritation process is around 2 mg/L Even when the anammox process is inhibited by influent DO temporarily, the inhibition is solved by decreasing the DO concentration

0.0 1.0 2.0 3.0 4.0 5.0

0.0 1.0 2.0 3.0 4.0 5.0

Time (day)

Fig 5 - Recovery from DO inhibition Nitrogen conversion rate of reactor 1(closed

circles), nitrogen conversion rate of reactor 2(closed squares) and the solid line (no symbol) shows influent DO concentration

ACKNOWLEDGEMENT

This study was supported by NEDO (New energy and industrial technology development organization), Japan

REFERENCES

Furukawa K., Lieu P K., Tokitoh H and Fujii T (2006) Development of single-stage nitrogen removal using anammox and partial nitritation (SNAP) and its treatment

performances, Wat Sci Technol., 53(6), 83-90

Furukawa K., Rouse J D., Bhatti Z I., Imajo U., Nakamura K and Ishida H (2002) Characterization of microbial community in an anaerobic ammonium-oxidizing

biofilm cultured on a nonwoven biomass carrier, J Biosci Bioeng, 94(5), 87-94

Hellinga C., Schellen A A J C., Mulder J W., Van Loosdrecht M C M and HeijnenWater J J (1998) The Sharon process: An innovative method for nitrogen

removal from ammonium-rich waste water, Science and Technol., 37(9), 135-142

Isaka K., Date Y., Sumino T and Tsuneda S (2007) Ammonium removal performance

of anaerobic ammonium-oxidizing bacteria immobilized in polyethylene glycol gel

carrier, Appl Microbiol Biotechno., 76(6), 1457-1495

Isaka K., Itokawa H., Kimura Y., Noto K., Murakami T and Sumino T (2009) Novel autotrophic nitrogen removal system using gel entrapment technology in Proceedings of 6th IWA leading edge conference on water and wastewater technologies, Singapore

Liu S., Yang F., Xue Y., Gong Z., Chen H., Wang T and Su Z (2008) Evaluation of oxygen adaptation and identification of functional bacteria composition for

anammox consortium in non-woven biological rotating contactor, Bioresource

Thechnol., 99(17), 8273-8279

Mulder A., Van de Graaf A A., Robertson L A and Kuenen J G (1995) Anaerobic

ammonium oxidation discovered in a denitrifying fluidized bed reactor, FEMS

Microbiol Ecol., 16(3), 177-184

Slinkers A O., Derwort N., Gomez J L C., Strous M., Kuenen J G and Jetten M S M (2002) Completely autotrophic nitrogen removal over nitrite in one single reactor,

Wat Res., 36, 2475-2482

Strous M., Heijnen J J., Kuenen J G and Jetten M S M (1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic

ammonium-oxidation microorganisms, Appl Microbiol Biotechnol., 50(5),

589-596

Sumino T., Noto T., Ogasawara T., Hashimoto N and Suwa Y (1997) Nitrification of high concentration ammonium nitrogen using immobilized nitrifying bacteria, WEFTEC 70th Annual Conference and Exposition, 165-172

Third K A., Slinkers A O., Kuenen J G and 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, System

Appl Microbiol., 24, 588-596

Tokutomi T (2004) Operation of a nitrite-type airlift reactor at low DO concentration,

Water Science and Technology, 49, 81-88

Van de Graaf A A., Peter de Bruijn, Robertson L A., Jetten M S M and Kuenen J G (1996) Autotrophic growth of anaerobic ammonium-oxidizing microorganisms in a

fluidized bed reactor, Microbiol., 142(8), 2187-2196

Van der Star W R L, Abma E R., Blommers D., Mulder J W., Tokutomi T., Strous M., Picioreanu C and Van der Loosderecht M.C.M (2007) Startup of reactors for anoxic ammonium oxidation: Experience from the first full-scale anammox reactor

in Rotterdam, Wat Res., 41, 4149-4163

Weatherburn M W (1967) Phenol-hypochlorite reaction for determination of ammonia,

Analytical Chemistry, 39, 971-974

Yamamoto T., Takaki K., Koyama T and Furukawa K (2008) Long-term stability of partial nitritation of swine wastewater digester liquor and its subsequent treatment

by Anammox, Bioresource Thechnol., 99(14), 6419-6425