Achieving Mainstream Nitrogen Removal through Coupling Anammox

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Achieving mainstream nitrogen removal through coupling anammox with denitratation Bin Ma, Wenting Qian, Chuansheng Yuan, Zhiguo Yuan, and Yongzhen Peng Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01866 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on June 29, 2017

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Environmental Science & Technology

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Achieving mainstream nitrogen removal through coupling anammox with denitratation

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Bin Ma1, Wenting Qian1, Chuansheng Yuan1, Zhiguo Yuan2, Yongzhen Peng1, *

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1. National Engineering Laboratory for Advanced Municipal Wastewater Treatment and

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Reuse Technology, Beijing University of Technology, Beijing, China;

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2. Advanced Water Management Center, The University of Queensland, St Lucia, QLD 4072,

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Australia.

1

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* Corresponding author: [email protected], +86-10-67392627

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ABSTRACT: Achieving maintream anammox is critical for energy-neutral sewage treatment.

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This study presents a new way to achieve mainstream anammox, which couples anammox

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with denitratation (nitrate reduction to nitrite) instead of nitritation (ammonium oxidation to

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nitrite). An anoxic/oxic (A/O) biofilm system treating systhetic domestic wastewater was

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used to demonstrate this concept for over 400 days. This A/O biofilm system achieved a total

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nitrogen (TN) removal efficiency of 80±4% from the influent with a low C/N ratio of 2.6

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and a TN concentration of 60.5 mg/L. Nitrogen removal via anammox was found to account

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for 70% of dinitrogen production in the anoxic reactor. Batch tests confirmed that the anoxic

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biofilm could oxidize ammonium using nitrite as electron acceptor, and that it had a higher

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nitrate reduction rate than the nitrite reduction rate, thus producing nitrite for the anammox

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reaction. Metagenomic analysis showed that Candidatus Jettenia caeni and Candidatus

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Kuenenia stuttgartiensis were the top two dominant species in anoxic biofilm. Genes

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involved in the metabolism of the anammox process were detected in anoxic biofilm. The

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abundance of nitrate reductase (73360 hits) was much higher than nitrite reductase (13114 1 / 33

ACS Paragon Plus Environment


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hits) in anoxic biofilm. This system can be easily integrated with the high-rate activated

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sludge technology, which produces an effluent with a low C/N ratio. While this new design

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consumes 21% more oxygen in comparison to the currently studied nitritation/anammox

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process, the nitrite-producing process appears to be more stable.

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Keywords: biological nitrogen removal; mainstream; anaerobic ammonium oxidation

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(anammox); denitratation; sewage;

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30 31

TOC Art

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INTRODUCTION

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Nitrogen needs to be removed from wastewater to control eutrophication of waterbodies.

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The traditional biological nitrogen removal technologies used in most wastewater treatment

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plants (WWTPs) involve nitrification and denitrification. Ammonium is firstly oxidized by

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ammonium oxidizing bacteria (AOB) to nitrite, which is further oxidized to nitrate by nitrite

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oxidizing bacteria (NOB) under aerobic condition. These steps require supply of oxygen

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through aeration, which comprises approximately 50% of energy consumption in WWTPs 1.

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Nitrate produced by nitrification is converted to nitrogen gas (and hence removed from

41

wastewater) through denitrification, which requires organic matter contained in wastewater,

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and sometimes also provided externally, as electron donor. This process substantially reduces

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the amount of organic carbon available for bioenergy recovery.

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Recently, anaerobic ammonium oxidizing (Anammox) bacteria have been discovered,

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initially in a denitrifying bed 2. These bacteria oxidize ammonium to nitrogen gas with nitrite

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as electron acceptor under anoxic condition 3. The application of anammox bacteria in

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wastewater treatment reduces the energy consumption of WWTPs since approximately 45%

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of the influent ammonium could be oxidized anoxically without the supply of oxygen 4.

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Anammox has been widely applied to the treatment of N-rich wastewater

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refered to as the side-stream anammox process. The application of anammox to mainstream

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treatment, typically referred to as the mainstream anammox process, has also been proposed,

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which is expected to enable energy-neutral or even energy-positive sewage treatment 7.

5, 6

, which is ofen

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Nitrite and ammonium are needed for the growth of anammox bacteria. While

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ammonium is directly available in sewage, nitrite needs to be produced in the treatment 3 / 33



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process. There are two main pathways to produce nitrite, namely nitritation (i.e. ammonium

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oxidation to nitrite) and denitratation (i.e. nitrate reduction to nitrite). Studies on the

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anammox-based mainstream treatment of sewage have mainly focussed on the

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nitritation/anammox pathway

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date.

8-12

, with the denitratation/anammox pathway unexplored to

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Figure 1

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However, several very recent studies have demonstarted that nitrite production could be

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stably achieved, with a nitrite accummulation rate of 80%, in a denitratation upflow sludge

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bed

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denitratation process has been used to reduce nitrate to nitrite in an anammox reactor treating

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N-rich wastewater 15, 16, in which nitrate produced by anammox was converted back to nitrite

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through the addition of an external carbon source.

13

. N2O production was low during the reduction of nitrate to nitrite

14

. Indeed, this

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In this study, we hypothesize that denitratation could potentially provide an alternative

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pathway to produce nitrite in a mainstream anammox system. The proposed conceptual

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process design is schematically shown in Fig. 1A. With this design, sewage containing

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ammonium and a low level of organic carbon (e.g. effluent from a high-rate activated sludge

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reactor17) is fed to the denitratation/anammox reactor, along with nitrate produced in the

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nitrification reactor. In the presence of anammox bacteria in the anoxic reactor to consume

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nitrite, denitrification could stop at nitrite, resulting in a partnership between denitrifiers and

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anammox bacteria that removes nitrogen through the denitratation/anammox pathway.

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This study aims to evaluate the feasibility of achieving mainstream anammox coupled

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with denitratation. A lab-scale A/O biofilm system fed with synthetic sewage-like wastewater 4 / 33


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was operated. The long-term performance of the A/O biofilm system was monitored over one

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year. The nitrogen removal pathways in the anoxic zone was investigated through measuring

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the biofilm activity in controlled batch tests under various process conditions, and assessing

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the biofilm microbial structure using metagenomic analysis. The robustness of the process

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was further investigated by monitoring the recovery process following a four-week starvation

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period.

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MATERIALS AND METHODS

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Reactor set-up and operation

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A laboratory A/O biofilm-based denitratation/anammox system implementing the

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conceptual process shown in Fig.1A is shown in Fig.1B. Two columnar reactors were used as

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the anoxic reactor and the aerobic reactor, respectively. A three-phase separator was installed

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at the top of each reactor to separate gas, water and sludge under high turbulence conditions.

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The total working volume of the anoxic reactor was 1.2 L with an internal diameter of 6 cm

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and a height of 43 cm. The total working volume of the aerobic reactor was 3 L with an

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internal diameter of 6 cm and a height of 108 cm.

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The anoxic reactor was innoculated with a mixture of flocular anammox sludge (0.4 g 18, 19

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MLSS) and denitrifying phosphorus removal sludge (1.5 g MLSS)

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volume) bare biofilm carriers (K1 bio-carriers) were added to the anoxic reactor at the same

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time. The K1 bio-carriers were made of polyethylene and shaped like a cylinder (10 mm

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diameter and 7 mm long). The carriers had an effective specific surface area of 500 m2/m3

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with a density of 0.98 g/cm3. The anoxic reactor had a net volume of 0.6 L for water after the 5 / 33


. 1.1 L (packing


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addition of K1 bio-carriers. The aerobic reactor was innoculated with 1.7 L (packing volume)

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mature K3 bio-carriers obtained form a pilot-scale nitrifying biofim reactor. K3 bio-carriers

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were made of polyethylene and shaped like a cylinder (25 mm diameter and 10 mm long)

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with an effective specific surface area of 350 m2/m3. The aerobic reactor had a net volume of

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2.4 L for water after the addition of K3 bio-carriers.

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The influent flow rate was 19.2 L/d, which was controlled by a peristaltic pump. The

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nitrate recirculation rate was controlled at 3 times that of influent (i.e. at 57.6 L/d), also by a

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peristaltic pump. These flow rates give a hydraulic time of 0.75 hr and 3 hr in the anoxic and

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aerobic reactor, respectively. The temperature in both reactors was controlled at 27 ± 1ºC

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with a thermostatic heater. A constant air flow rate was applied in the aerobic reactor to

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supply enough oxygen for nitrifiers. The influent and effluent pH and DO concentrations of

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two reactors were measured using the Handheld, 3420 Multi-Parameter Meter (WTW Company).

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Neither pH nor DO was controlled. However, the constant air flow rate applied was adequate

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to ensure a DO concentration in the range of 7-9 mg/L.

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The synthetic watewater used in the study comprised various components to simulate

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domestic wastewater, with the chemical oxygen demand (COD) and NH4+-N concentrations

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being approximately 160 mg/L and 60 mg/L, respectively. The detailed composition was (per

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litre): 205mg CH3COONa, 225 mg NH4Cl (60 mg NH +4 -N), 18 mg KH2PO4, 90 mg

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MgSO4⋅7H2O, 900 mg NaHCO3, 14 mg CaCl2⋅2H2O, and 1.25 mL trace element solution A

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and B. Trace element solution A contained (per litre): 5 g EDTA and 5 g FeSO4; and trace

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element solution B contained (per litre): 15 g EDTA, 0.43 g ZnSO4⋅7H2O, 0.99 g

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MnCl2⋅4H2O, 0.25 g CuSO4⋅5H2O, 0.22 g NaMoO4⋅2H2O, 0.19 g NiCl2⋅6H2O, and 0.014 g 6 / 33


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H3BO4.

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The system was operated for 408 days. In order to demonstrate the robustness of this

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A/O biofilm process, a 28-day starvation period was applied (Day 264 to Day 292). In this

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period, the influent, air supply to the aerobic reactor and recirculation from the aerobic

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reactor to the anoxic reactor were all stopped.

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Batch tests to determine the activity of the anoxic biofilm

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After steady operation of the A/O biofilm system was achieved, batch tests were carried

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out to investigate the activity of the anoxic biofilm. Biofilm carriers of 500 mL were taken

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from the anoxic reactor and transferred to an Erlenmeyer flask (1L). The flask was sparged

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with nitrogen to remove any residual oxygen. The flask was then sealed with a rubber stopper

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to maintain anaerobic condition. In Batch Test A, a stock solution containing ammonium

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(38.21 g/L NH4Cl), nitrate (60.83 g/L NaNO3) and sodium acetate (12.81 g/L CH3COONa)

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was added at the beginning with a COD/NO3--N ratio of 2.5. In Batch Test B, a stock solution

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containing ammonium (38.21 g/L NH4Cl) was added at the beginning. In this test, a nitrite

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stock solution (4.93 g/L NaNO2) was step-wise added in three pulses, each theorectically

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raising the nitrite concentration by 1.0 mgN/L. In Batch Test C, a stock solution containing

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nitrate (60.83 g/L NaNO3) and sodium acetate (12.81 g/L CH3COONa) was added at the

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beginning with a COD/NO3--N ratio of 2.5. All the tests were carried out at a temperature of

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27 ± 1ºC, with the magnetic stirrers kept at 100 rpm for completely mixing. During the batch

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tests, water samples were taken at an interval of 30 min for the analysis ammonium, nitrite

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and nitrate. Test A and Test C were run for 180 min, with 7 samples taken. Test B was run for

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300 min with 11 samples taken. 7 / 33



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Sampling and analytical methods

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Wastewater samples were collected, three times per week, from the influent, and effluent

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from the anoxic and aerobic reactors. Water samples were taken using a syringe and

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immediately filtered through disposable Millipore units (0.22 µm Pore size) for the analyses

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of ammonium, nitrite, nitrate, and soluble chemical oxygen demand (SCOD), according to

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standard methods

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measurement at a ratio of 1.1gSCOD/g NO2--N. The dissolved oxygen (DO) concentration,

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pH and temperature were measured with oxygen and pH probes (WTW 3420, WTW

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Company). A biofilm sample was collected from the anoxic reactor on Day 401 to investigate

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the microbial community in the anoxic biofilm.

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Shotgun metagenomic sequencing and bioinformatic analysis

20

. The SCOD data were corrected because nitrite contributes to SCOD

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The microbial community in the anoxic biofilm was analyzed using shotgun

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metagenomic sequencing and bioinformatic analysis. DNA was extracted from the anoxic

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biofilm sample in duplicate using the FastDNA SPIN Kit for Soil (QBIOgene, Carlsbad, CA,

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USA) according to the manufacturer's protocol. The integrity of the DNA was verified using

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gel electrophoresis. Illumina shotgun DNA library construction and sequencing was

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conducted by the Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China).

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After fragmentation, pairedend fragment library in length of ∼300 bp was constructed.

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Sequencing was performed using Illumina Hiseq 4000 (Illumina, USA). Raw reads (150 bp

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in length) were trimmed to remove low quality reads that contained ambiguous nucleotides,

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shorter than 50 bp, or had a quality value lower than 20. In total, 56381196 clean reads were

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generated. The sequences were deposited in NCBI Sequence Read Archive (SRA) with 8 / 33


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accession number SRP100137. After quality filtering described above, the cleaned sequence reads were assembled into

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contigs using SOAPDENOVO ASSEMBLER (v 1.06) 21. A range of k-mer size values (39

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47) were investigated. Only contigs longer than 500 bp were used for further analysis. The

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successfully aligned reads were assigned as ‘assembled reads’.

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−

Open reading frames (ORFs) were predicted from contigs using METAGENE using 22

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default settings

. The predicted ORFs longer than 100 nt were translated into protein

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sequences based on the NCBI translation table 11

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‘redundant’ (or highly similar) sequences and to determine gene abundance and statistics

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among the samples

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mapped back to the non-redundant ORF set for each sample and the coverage for each ORF

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was calculated as the number of mapped reads.

24

23

. CD-HIT was then used to remove

. All of the predicted ORFs were imported into CD-HIT. Reads were

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To predict the phylogenetic origins of the functional genes, their protein sequences were

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searched against the NCBI NR database using BLASTP (version 2.2.28+) 25. Sequences were

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assigned to NCBI taxonomies with MEGAN by using the lowest common ancestor algorithm

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and the default parameters. The relative abundance of each gene was determined by

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calculating the percentage of hits number of the gene to the total number of the sequencing

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reads of the biofilm sample. Functional annotation and taxonomic analysis of the key

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enzymes for denitrification and anammox were done as described by Guo, et al. 26.

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RESULTS

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Nitrogen removal performance of the A/O biofilm system 9 / 33



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Ammonium was efficiently removed with an average NH4+-N removal efficency of 98%

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during the whole study of 408 days (Fig.2A). High ammonium removal performance was

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achieved immediately at the beginning of the reactor operation due to the seed of nitrifying

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biofilm carriers, which adapted quickly to the new operational conditions. There were nitrate

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and nitrite in the effluent of the anoxic reactor during most of the operational time (Fig.2 B,

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C). This was likely related to the low supply of organic matter for denitrification (Fig.3).

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Nitrite and ammonium co-existed in the anoxic reactor, which created conditions for the

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growth of anammox bacteria. During the stable operation stage (between Day 293 and Day

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408), the average nitrate concentrations were 2.4 and 11.3 mgN/L in the effluent of the anoxic

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and aerobic reactors, respectively (Fig.2 C). Therefore, there was a nitrate production of 8.9

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mgN/L in the aerobic reactor. The TN removal performance gradually increased from day 1

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to day 130, and then remained stable with a TN removal efficiency of 73±8% between Day

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130 and Day 263 (Fig.3B). In order to demonstrate the robustness of this A/O biofilm process,

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the recovery of nitrogen removal performance after a 28 day (Day 264 to Day 292) starvation

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period was investigated. The TN removal efficiency quickly improved to over 80% within

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one day (Day 293). This indicated that the A/O biofilm process was robust. During the last

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115 days after the starvation, an TN removal efficiency of 80±4% was achieved, when the

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influent TN and C/N were 60.5±5.7 mgN/L and 2.6±0.5, respectively (Fig.2D, Fig.3B).

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Figure 2

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Figure 3

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Mass balance analysis of the influent and effluent nitrogen data of the anoxic reactor

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(see Supplementary Information for equations) in the stable operation period (between Day 10 / 33


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293 and Day 408) showed that 92% of TN removed in anoxic reactor was converted to

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dinitrogen, with the rest assimilated into biomass. Importantly, 70% of dinitrogen produced in

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this reactor was by the anammox bacteria.

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Activity of the anoxic biofilm

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TN removal in the anoxic reactor was primarily responsible for the TN removal in the

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A/O system, accounting for 73% of the TN removal in the 115 days after starvation. In order

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to investigate the nitrogen removal pathway in the anoxic reactor, batch tests were conducted

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with the biofilm obtained from the anoxic reactor. In Batch Test A, which simulated the

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condition of the anoxic reactor, nitrate, ammonium and organic matter were added

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simultaneously at the beginning of the anoxic test. Nitrogen balance was performed based on

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the nitrogen conversion data in the first 60 min (Fig.4A), which showed that the denitratation

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and anammox reactions occurred simultaneously. Of the 5.33 mg/L TN removal (Fig.4A), 92%

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was estimated to be converted into dinitrogen with the remaining converted to biomass. Of

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the estimated dinitrogen production, 73% was estimated to be produced by anammox bacteria,

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with the remainder attributed to denitrifiers. The estimated anammox contribution to TN

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removal is very close to that (70%) estimated from the long-term operation data of the anoxic

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reactor. During the last 90 minutes, nitrate, ammonium and nitrite remained approximately

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constant due to the depletion of acetate leading to cessasion of denitratation (Fig. 4A). In

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order to confirm the occurance of the anammox reaction, Batch Test B was conducted with

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the addition of nitrite and ammonium without acetate. During the 300 min operation, NH4+-N

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and NO2--N cwere reduced by 2.01 mg/L and 2.10mg/L, respectively, and NO3--N increased

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by 0.42 mg/L (Fig.4B). The ratio of nitrite and ammonium consumption was 1.05, and the 11 / 33



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ratio of nitrate production to ammonium consumption was 0.21. These two ratios are in line

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with the stoichiometric values of 1.32 and 0.26, respectively, for the anammox reaction (see

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Equation 1 in Supplementary Information). In order to confirm nitrite production by

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denitratation, Batch Test C was conducted with the addition of nitrate and acetate. In the first

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30 min, nitrate was reduced by 5.36 mg/L (Fig.4C), and nitrite concentration increased by

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2.23 mg/L, which accounted for 42% of nitrate reduced. The removal of 58% of the nitrite

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produced through denitratation by denitrifier was slightly higher than the value of 28%

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obtained in Batch Test A, where denitratation and anammox reaction occurred simultaneously.

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Anammox competed for nitrite with denitrifiers, resulting in a decrease in nitrite consumption

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by denitrifiers. Figure 4

239 240

Microbial community in the anoxic biofilm

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The microbial community in the anoxic biofilm was analysed through metagenomic

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sequencing. To date, anammox bacteria have been divided into six genera: Candidatus

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Kuenenia, Candidatus Brocadia, Candidatus Jettenia, Candidatus Anammoxoglobus,

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Candidatus Scalindua, and Candidatus Anammoximicrobium Kuenen

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Candidatus Jettenia (17.83%) and Candidatus Kuenenia (2.62%) were detected in the anoxic

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biofilm (Fig.5A), with Candidatus Jettenia caeni and Candidatus Kuenenia stuttgartiensis

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being the top two dominant species in anoxic biofilm (Fig.5B). Thauera was the second most

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abundant genus in the anoxic biofilm at 5.27% (Fig.5A).

27, 28, 29

. In this study,

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Figure 5

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To better understand the functional diversity, especially the nitrogen metabolism 12 / 33


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pathways in the anoxic biofilm, we analyzed the abundance and the taxonomic origins of the

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key enzymes for denitrification and anammox (Fig.6). The hydrazine dehydrogenase gene

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(Hdh) and the hydrazine synthase gene (Hzs), which are the key functional genes of

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anammox bacteria, were detected in the anoxic biofilm. These two genes were mostly

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associated with Candidatus Jettenia and Candidatus Kuenenia (Fig.6). It’s interesting that the

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abundance of nitrate reductase (Nar) (73360 hits) was much higher than that of the nitrite

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reductase (Nir) (13114 hits) in the anoxic biofilm. Further, most of Nar (25596 hits) and Nir

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(6522 hits) were associated with Candidatus Jettenia. It was also found that Thauera, the

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second most abundant genus in the anoxic biofilm after the anammox genus of Candidatus

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Jettenia, contained much more Nar (5892 hits) than Nir (588 hits) (Fig.6). This would lead to

261

a higher nitrate reduction rate and a lower nitrite reduction rate, potentially resulting in nitrite

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accumulation for anammox consumption. Therefore, Thauera may play an important role in

263

providing nitrite for anammox bacteria through reducing nitrate to nitrite in the anoxic reactor.

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This finding agrees with the phenomenon of nitrite accumulation in Batch Test C (Fig.4C).

265

These results indicate that the anoxic biofilm had the potential of combining denitratation

266

with anammox. Figure 6

267

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DISCUSSION

269

Mechanism for achieving mainstream anammox coupled with denitratation

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This study reveals for the first time that it is feasible to remove nitrogen from

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sewage-like wastewater by coupling anammox with denitratation in an A/O biofilm system.

272

The premise of achieving anammox coupled with denitratation is a stable supply of nitrite to 13 / 33



273

anammox bacteria. The batch tests showed that the anoxic biofilm had a higher nitrate

274

reduction rate than nitrite reduction rate (Fig.4C), resulting in nitrite accumulation during the

275

denitrification process, feeding the anammox bacteria. Thauera was the second most

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abiundant genus in the anoxic biofilm after the anammox genus of Candidatus Jettenia

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(Fig.5A). Thauera was reported to have the ability to reduce NOx--N heterotrophically.

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Transient accumulation of nitrite was previously reported in nitrate reduction by Thauera

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phenylacetica30. Du, et al. 31 found that Thauera accounted for 67.25 % of microorganisms in

280

a denitrifying reactor with a peak nitrite accumulation rate of 90%. In their study, it was

281

observed that the specific nitrate reduction rate (59.61-82.31mg N/g VSS/h) was much higher

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than the specific nitrite reduction rate (7.30-8.80 mg N/g VSS/h) in the presence of nitrate.

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When nitrate was completely reduced, the specific nitrite reduction rate increased to

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22.70-31.96 mg N/g VSS/h, which was approximately three times the value observed in the

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presence of nitrate31. This indicated that the denitrifiers dominated by Thauera preferred to

286

use nitrate over nitrite as electron acceptor, which was possibly caused by the lower

287

competitive capability of Nir than Nar for electrons. This hypothesis is supported by our

288

results that Thauera, the dominant denitrifier in the anoxic biofilm, contained much more Nar

289

(5892 hits) than Nir (588 hits) (Fig.6). Therefore, Thauera may have played an important role

290

in providing nitrite for anammox bacteria through reducing nitrate to nitrite in the anoxic

291

reactor.

292

The limitation in the organic matter supply likely benefited the stable supply of nitrite to

293

anammox bacteria through denitratation. Nitrite could be reduced by the denitrifiers

294

dominated by Thauera, and the specific nitrite reduction rate increases after the depletion of 14 / 33


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nitrate when organic matter is sufficient31. Therefore, in order to accumulate more nitrite for

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anammox, organic matter should be completely consumed before the depletion of nitrate,

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which was achieved due to the low C/N wastewater used in our study. Oh and Silverstein

298

found that the organic matter oxidation rate was not affected by the C/N ratio. However, the

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flow of substrate-derived electrons between nitrate and nitrite reduction changed from

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balanced distribution to an imbalanced one favoring nitrate reduction resulting in nitrite

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accumulation when the C/N ratio decresed 32. Nitrite accumulation during denitrification with

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limited carbon was also observed in several other studies31, 33-35. In this study the influent

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with a COD/TN ratio of 2.6 did not provide an adequate amount of organic carbon for full

304

denitrification, since a theorectical COD/TN ratio of 4.1 is required for complete

305

denitrification (see Equation 2 and 3 in Supplementary Information). This low COD/TN ratio

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in influent likely contributed to nitrite accumulation during denitrification, which was of

307

benefit to the growth of anammox bacteria.

32

308

Nitrite accumulation during denitrification would also be affected by the type of carbon

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sources. van Rijn, et al. 36 found more nitrite accumulation when acetate or propionate served

310

as electron donor than when butyrate, valerate or caproate was electron donors. Compared to

311

acetate or methanol, glucose resulted in more nitrite accumulation during the nitrate reduction

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achieved by mixed activated sludge

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accmulated during the reduction of nitrate using glucose as electron donor, and the nitrite

314

accumulation peak was observed when aceate was used as carbon source. This difference

315

may be related to the bacterial community. Acetate was used as the organic matter in this

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work, this may have favoured nitrite accumulation during denitrification. However, nitrite

34

. In contrast, Sun, et al.

15 / 33


37

observed that no nitrite


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accumulation was reported in the anoxic zone of a pilot-scale modified UCT step feed

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biological nitrogen removal process treating real municipal wastewater, and this phenomenon

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lasted for more than one month in the WWTP when the water temperture was above 25ºC 34.

320

Based on this report, denitratation could be achieved in the anoxic reactor when the A/O

321

biofilm system is used to treated real municipal wastewater. However, more experimental

322

studies are required to verify the performance of the proposed process for real sewage

323

treatment.

324

Another important factor for achieving anammox coupled with denitratation is the

325

enrichment of anammox bacteria. In this study, forming biofilm was used to reach a good

326

retention of anammox bacteria. In steady state, the anoxic biofilm contained Candidatus

327

Jettenia (17.83%) and Candidatus Kuenenia (2.62%) as the dominating anammox bacteria.

328

Nitrogen removal via anammox was found to account for 70% of dinitrogen production in the

329

anoxic reactor. This reveals that anammox bacteria could coexist with heterotrophic

330

denitrifiers in the anoxic reactor fed with wastewater contained organic matter. It has been

331

suggested that organic matter decreased anammox activity

332

has been updated by more recent studies showing that anammox bacteria can survive in the

333

presence of organic substrates39,

334

Interestingly, Candidatus Jettenia caeni, the most abundant anammox species in our reactor,

335

could reduce nitrate to nitrite or ammonium couple to the oxidation of acetate 29. Futhermore,

336

it has been reported that a low level of organic matter enhanced nitrogen removal in an

337

anammox eractor through the reduction of nitrate produced by anammox to nitrite. However,

338

it has also been reported that further increased organic loading rate could negatively affect the

40

38

. However, this understanding

and can in fact oxidize acetate and propionate

16 / 33


41-43

.

Page 17 of 33


339

anammox process by facilitating heterotrophic denitrification40, 44. This study reveals that a

340

COD/TN ratio of 2.6 is suitable for anammox growth.

341

Implication of the findings

342

This study proposed a new approach to achieve mainstream anammox by coupling it

343

with denitratation. A good nitrogen removal performance of 80% and a strong stability

344

towards starvation were achieved in the A/O biofilm system using this strategy. This system

345

can be easily integrated with the high-rate activated sludge technology, which produces an

346

effluent with a low C/N ratio and harvests organic matter into sludge for biogas production.

347

Rahman, Meerburg, Ravadagundhi, Wett, Jimenez, Bott, Al-Omari, Riffat, Murthy and De

348

Clippeleir 17 compared conventional high-rate activated sludge system (including continuous

349

stirred reactor and plug flow reactor) with the high-rate contact-stabilization (CS) process,

350

and concluded that a better organic matter recovery was achieved with the latter process. The

351

soluble COD to NH4+-N ratio was 2.2-2.8 in the high-rate CS process 17. As revelaed in our

352

study, this effluent contains enough organic matter for denitratation in the AO biofilm system.

353

Compared with the currently studied nitritation/anammox pathway, which theoretically

354

require no COD, this denitratation/anammox strategy need organic matter for denitratation

355

with 1.05 mg COD / mg N (Table 1, Fig.S1 in Supplementary Information). However, the

356

organic matter presented in the effluent of a high–rate activated sludge system is adequte to

357

meet this requirement

358

denitratation/anammox strategy. The denitratation/anammox process is estimated to consume

359

21% more oxygen than the nitritation/anammox process (Table 1), which incur additional

360

costs. However, the denitratation/anammox process may be more robust. During the over 400

17

. Therefore, organic matter is not a problem for application of this

17 / 33



361

day study, the system displayed stable nitrogen removal performace after the initial transient

362

period. This is in clear contrast to the mainstream nitritation/anammox process, which is

363

often unstable due to the growth of nitrite-oxidising bacteria 10, 45.

364

Table 1

365

The feasibility of achieving anammox coupled with denitratation in the mainstream

366

treatment of sewage-like wastewater was demonstrated through a long-term laboratory study.

367

The main conclusions are: Nitrogen removal through coupling the anammox and

368

denitratation reactions can be achieved in a A/O biofilm system. The anammox/denitratation

369

process can effectively remove nitrogen from wastewater with low C/N ratios.The strategy

370

demonstrated in this study has the potential to be integrated with the high-rate activated

371

sludge technology to enable maximum energy recovery while achieving satisfactory nitrogen

372

removal.

373

ASSOCIATED CONTENT

374

Supporting Information

375

Additional details of results are included. Determining anammox contribution to total

376

nitrogen removal (Text S1), Comparision of the processes of nitritation/anammox and

377

denitratation/anammox (Text S2), and Nitrogen conversion in the processes of

378

nitritation/anammox and denitratation/anammox (Fig. S1).

379

AUTHOR INFORMATION

380

Corresponding Authors

381

*(Y.P.) Phone: 86-10-67392627; E-mail: [email protected].

382

ORCID 18 / 33


Page 18 of 33

Page 19 of 33


383

Yongzhen Peng: 0000-0002-0826-8613

384

Notes

385

The authors declare no competing financial interest.

386

ACKNOWLEDGEMENTS

387

This research was financially supported by the National Natural Science Foundation of

388

China (51508008 and 51478013), Training Programme Foundation for the Talents in Beijing

389

(2014000020124G043) and the 111 Project.

390

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Jetten, M. S. M.; Strous, M., Candidatus 'Brocadia fulgida': an autofluorescent anaerobic

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522

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10199-10210.

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524

Table and Figure captions

525

Table 1 Comparision of Denitratation/anammox and Nitritation/anammox processes

526

Fig.1 Schematic of a biofilm-based denitratation/anammox process (A), the A/O biofilm

527 528

system used in the experimental study (B) . Fig.2 Nitrogen concentration in the influent and effluent of the A/O biofilm process, the

529

influent (Inf.), anoxic reactor effluent (An.) and aerobic reactor effluent (Eff.) NH4+-N,

530

NO2--N, NO3--N, and TN concentrations. The system was starved between Day 264 and

531

Day 292.

532

Fig.3 A: COD concentrations of the influent (Inf.), anoxic reactor effluent (An.) and aerobic

533

reactor effluent (Eff.) of the A/O biofilm process, B: variations of the influent COD/TN

534

ratio (Inf. C/N) and TN removal efficiencies. The system was starved between Day 264

535

and Day 292.

536

Fig.4 Variations of nitrogen concentrations during the three batch tests. A: ammonium, nitrate

537

and organic matter were added at the beginning with a COD/NO3--N ratio of 2.5; B:

538

ammonium was added at the beginning, and nitrite was stepwise fed in three pulses; C:

539

nitrate and organic matter were added at the beginning with a COD/NO3--N ratio of 2.5.

540 541

Fig.5 Abundance of the major bacterial genera (A) and species (B) (>1.0%) in the anoxic biofilm.

542

Fig.6 Taxonomic origins of the key enzymes in nitrogen metabolism detected in the anoxic

543

biofilm reactor. Hdh, hydrazine dehydrogenase; Hzs, hydrazine synthase; Nar, nitrate

544

reductase; Nir, nitrite reductase; Nor, nitric oxide reductase; Nos, nitrous oxide

545

reductase; and Nrf, nitrite reductase (ammonia-forming). 26 / 33


Page 26 of 33

Page 27 of 33

546


Table 1 Comparision of Denitratation/anammox and Nitritation/anammox processes Organic matter demand

Oxygen demand

(mg COD / mg N )

(mg DO / mg N)

Denitratation/anammox

1.05

2.28

Nitritation/anammox

0.00

1.89

Process

547

27 / 33



548 549 550

Fig.1 Schematic of a biofilm-based denitratation/anammox process (A), the A/O biofilm system used in the experimental study (B).

551

28 / 33


Page 28 of 33


80 70 60 50 40 30 20 10 0 0

-

NO2 -N (mg/L)

+

NH4 -N (mg/L)

Page 29 of 33

8 7 6 5 4 3 2 1 0 0

Inf. NH4

A

50

100

150 Inf.NO2

B

200 250 300 Time(day) An.NO2 Eff.NO2

350

400

Starvation period

50

100

150 Inf.NO3

C

200 250 Time(day) An.NO3

300

350

400

Eff.NO3 Starvation period

30 20

-

NO3 -N (mg/L)

Eff.NH4

Starvation period

50 40

An.NH4

10

TN (mg/L)

0 0

552

80 70 60 50 40 30 20 10 0 0

50

100

150 Inf.TN

D

200 250 Time (day) An.TN

300

350

400

Eff.TN

Starvation period

50

100

150

200 250 Time(day)

300

350

400

553

Fig.2 Nitrogen concentration in the influent and effluent of the A/O biofilm process, the

554

influent (Inf.), anoxic reactor effluent (An.) and aerobic reactor effluent (Eff.) NH4+-N,

555

NO2--N, NO3--N, and TN concentrations. The system was starved between Day 264 and Day

556

292.

557

29 / 33



250

A

Inf.COD

An.COD

Page 30 of 33

Eff.COD

COD (mg/L)

200 150 100

8 7 6 5 4 3 2 1 0

0

50

100

150

200 250 Time(day)

350

400 1.0

RE. TN

Inf. C/N

B

300

0.8 Starvation period

0.6 0.4

RE. TN

Inf. C/N

0

558

Starvation period

50

0.2 0

50

100

150

200 250 Time(day)

300

350

400

0.0

559

Fig.3 A: COD concentrations of the influent (Inf.), anoxic reactor effluent (An.) and aerobic

560

reactor effluent (Eff.) of the A/O biofilm process, B: variations of the influent COD/TN ratio

561

(Inf. C/N) and TN removal efficiencies. The system was starved between Day 264 and Day

562

292.

563

30 / 33



Nitrogen concentration(mg/L)



Page 31 of 33

16 14 12 10 8 6 4 2 0

NH4

0

30

12

NH4

NO3

90 120 Time(min) NO2

A

TN

150 NO3

180

B

TN

10 8 6

Adding nitrite

4

Adding nitrite

Adding nitrite

2 0 0

30

60

10

564

60

NO2

90 120 150 180 210 240 270 300 Time(min) NO2

NO3

TN

C

8 6 4 2 0 0

30

60

90 120 Time(min)

150

180

565

Fig.4 Variations of nitrogen concentrations during the three batch tests. A: ammonium, nitrate

566

and organic matter were added at the beginning with a COD/NO3--N ratio of 2.5; B:

567

ammonium was added at the beginning, and nitrite was stepwise fed in three pulses; C: nitrate

568

and organic matter were added at the beginning with a COD/NO3--N ratio of 2.5.

569

31 / 33



A

570

B

571 572 573

Fig.5 Abundance of the major bacterial genera (A) and species (B) (>1.0%) in the anoxic biofilm.

574

32 / 33


Page 32 of 33

Page 33 of 33


575 576

Fig.6 Taxonomic origins of the key enzymes in nitrogen metabolism detected in the anoxic

577

biofilm reactor. Hdh, hydrazine dehydrogenase; Hzs, hydrazine synthase; Nar, nitrate

578

reductase; Nir, nitrite reductase; Nor, nitric oxide reductase; Nos, nitrous oxide reductase; and

579

Nrf, dissimilatory nitrite reductase.

33 / 33


Achieving Mainstream Nitrogen Removal through Coupling Anammox

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