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Complete Nitrogen Removal from Synthetic Anaerobic Sludge Digestion Liquor through Integrating Anammox and Denitrifying Anaerobic Methane Oxidation in a Membrane Biofilm Reactor Guo-Jun Xie, Chen Cai, Shihu Hu, and Zhiguo Yuan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04500 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 20, 2016

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Environmental Science & Technology

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Complete Nitrogen Removal from Synthetic Anaerobic Sludge Digestion Liquor

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through Integrating Anammox and Denitrifying Anaerobic Methane Oxidation in a

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Membrane Biofilm Reactor

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Guo-Jun Xie†, Chen Cai†, Shihu Hu† and Zhiguo Yuan*,†

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†

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QLD 4072, Australia

Advanced Water Management Centre, The University of Queensland, St Lucia, Brisbane,

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*Corresponding author.

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E-mail: [email protected];

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Tel: +61 (0) 7 336 54374;

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Fax: +61 (0) 7 336 54726.

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ACS Paragon Plus Environment


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Abstract

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Partial nitritation and Anammox processes are increasingly used for nitrogen

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removal from anaerobic sludge digestion liquor. However, their nitrogen removal

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efficiency is often limited due to the production of nitrate by the Anammox reaction

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and the sensitivity to the nitrite to ammonium ratio. This work develops and

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demonstrates an innovative process that achieves complete nitrogen removal from

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partially nitrified anaerobic sludge digestion liquor through the use of a membrane

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biofilm reactor (MBfR), with methane supplied through hollow fibre membranes.

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When steady state with a hydraulic retention time (HRT) of 1 day was reached, the

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process achieved complete nitrite and ammonium removal at rates of 560 mg N/L/d

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and 470 mg N/L/d, respectively, without any nitrate accumulation. The process is

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relatively insensitive to the nitrite to ammonium ratio, achieving complete nitrogen

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removal when their ratio in influent varied in the range of 1.125–1.32. Pyrosequencing

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and fluorescence in situ hybridization analysis revealed that denitrifying anaerobic

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methane oxidation (DAMO) archaea, Anammox bacteria and DAMO bacteria jointly

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dominated the microbial community. Mass balance analysis showed that nitrate

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produced by Anammox (122.2 mg N/L/d) was entirely converted to nitrite by DAMO

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archaea, while nitrite in the feed and produced by DAMO archaea was jointly removed

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by Anammox (90%) and DAMO bacteria (10%). The nitrogen removal rate of over 1

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kg N/m3/d is comparable to the practical rates reported for side-stream nitrogen

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removal processes.

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Keywords: Nitrogen removal; Denitrifying anaerobic methane oxidation; Membrane Biofilm

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Reactor; Anammox

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TOC Art

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1. Introduction

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Human activities have resulted in ever increasing discharge of municipal and

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industrial wastewater containing reactive nitrogen to the natural water bodies, which

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has contributed to a host of environmental problems, including eutrophication1,

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biodiversity loss2 and dysfunction of aquatic ecosystems3. Biological nitrogen removal

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is a critical process returning reactive nitrogen to the atmosphere as dinitrogen gas and

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maintaining the balance of the global nitrogen budget. However, conventional

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biological nitrogen removal processes require considerably energy to provide oxygen

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for aerobic conversion of ammonium to nitrate/nitrite by nitrification and then

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consumes large amounts of carbon sources for anaerobic conversion of nitrate/nitrite

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to nitrogen gas by heterotrophic denitrification4. As a result, traditional nitrification-

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denitrification process incurs significant operation costs. Achieving complete nitrogen

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removal from wastewater while minimizing energy and organic carbon consumption is

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a crucial challenge for the water industry.

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Anaerobic ammonium oxidation (Anammox) has the unique metabolic ability to

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combine ammonium and nitrite to form nitrogen gas5. Autotrophic nitrogen removal

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over nitrite can be achieved through combination of partial nitritation and Anammox,

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which entails significant advantages over classical nitrification/denitrification.

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Through partial nitritation, ammonium oxidizing bacteria (AOB) consumes a limited

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amount of oxygen to partially oxidize ammonium to nitrite6, while Anammox converts

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residual ammonia and nitrite into nitrogen gas under anoxic conditions. The

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combination of AOB and Anammox could save 60% of aeration and 100% of organic

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carbon, and reduce sludge production by 90%4. However, Anammox process faces the

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challenges that a strict nitrite to ammonium ratio of 1.32 is required7, and that

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Anammox converts 20% of the nitrite to nitrate (Reaction 1). This process (partial 4


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nitritation/Anammox) does not remove nitrate present in the wastewater, and hence its

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theoretical maximum nitrogen removal efficiency is 89%. In actual operation, the ratio

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of nitrite to ammonium may deviate significantly from the desired ratio, which often

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results in nitrogen removal efficiency about 70%8-10.

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NH 4+ + 1.32 NO2− → 1.02 N 2 + 0.26 NO3− + 2.03H 2O

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The discovery of denitrifying anaerobic methane oxidation (DAMO) process, in

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which methane is oxidized anaerobically to provide electrons for denitrification, not

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only forms an important link between global carbon and nitrogen cycles11-13, but also

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provides an opportunity for enhancing the nitrogen removal performance of the

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Anammox process using biogas methane as a cheap, supplementary electron donor14.

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A microbial consortium enriched from freshwater sediments was demonstrated to

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oxidize methane to carbon dioxide coupled to denitrification under strictly anaerobic

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conditions15. This consortium consisted of two distinct microorganisms, DAMO

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bacteria and DAMO archaea. DAMO bacteria convert nitrite to nitric oxide and then

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oxidize methane using intracellular oxygen produced from the dismutation of nitric

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oxide (Reaction 2)16, while DAMO archaea reduce nitrate to nitrite using electrons

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derived from methane oxidation through reverse methanogenesis (Reaction 3)17.

(1)

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3CH 4 + 8 NO2− + 8 H + → 3CO2 + 4 N 2 + 10 H 2O

(2)

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CH 4 + 4 NO3− → CO2 + 4 NO2− + 2 H 2O

(3)

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Nitrate reduction by DAMO archaea provides a novel possibility of achieving complete

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nitrogen removal through cooperation of Anammox and DAMO archaea. In such a process,

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nitrite and ammonium are converted into dinitrogen by Anammox with nitrate as a by-

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product (Reaction 1); methane is used by DAMO archaea to denitrify nitrate into nitrite

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(Reaction 3), which is subsequently removed by Anammox. The net reaction becomes

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Reaction 4, with a theoretical nitrite to ammonium ratio of 1.06 (from the Anammox reaction

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ratio of 1.32).

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NH 4+ + 1.06 NO2− + 0.065CH 4 → 1.02 N 2 + 2.16 H 2O + 0.065CO2

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In addition, the co-occurrence of nitrite and methane in this process could enable the

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growth of DAMO bacteria (Reaction 2), which is also responsible for nitrogen removal

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through direct conversion of nitrite to nitrogen gas. This reaction, if occurs, provides an

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additional degree of freedom making the process more flexible in terms of the nitrite to

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ammonium ratio required. As a result, complete nitrogen removal could be achieved by

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(4)

Anammox/DAMO without the requirement of the fixed ratio between nitrite and ammonium. The recent studies not only detected the co-occurrence of Anammox and DAMO

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organisms in natural environments, such as wetland18 and paddy soil19, 20, but also enriched in

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laboratory reactors fed with methane, ammonium and nitrate/nitrite17, 21, 22. However, the

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current nitrate/nitrite removal rates of suspended DAMO microorganisms were only 13-40.32

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mg N/L/d14, 21-24, which are too low for practical applications. In order to achieve a higher

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nitrogen removal rate, a membrane biofilm reactor (MBfR) was developed to deliver methane

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to the DAMO microorganisms in biofilm that grew on the outer surface of hollow fibre

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membrane25. To stimulate the growth of DAMO archaea and Anammox bacteria, nitrate and

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ammonium were provided in the feed. The nitrate removal rate by DAMO archaea was

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achieved at 190 mg N/L/d25 in sequencing batch operation, which was further enhanced to

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684 mg N/L/d through continuous operation26. However, the reactor was fed with nitrate

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(600–1500 mg N/L) and ammonium (300–600 mg N/L), which does not represent effluent

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from a partial nitritation reactor. Also, the reactor displayed poor and unstable nitrogen

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removal performance with the effluent nitrate and ammonium concentrations up to 300 mg

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N/L and 400 mg N/L, respectively.

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Therefore, the key objectives of this work are: (1) to develop the above-mentioned MBfR

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concept into a technology that removes nitrogen from partial nitritation effluent of anaerobic

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sludge digestion liquor; (2) to determine the effluent quality and nitrogen removal rate that

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are achievable with the MBfR technology; (3) to investigate the nitrogen removal by MBfR

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system when the nitrite to ammonium ratio varies. To achieve the above research objectives,

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a laboratory reactor was equipped with polypropylene hollow-fibres. The MBfR was fed with

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synthetic wastewater containing nitrite and ammonium mimicking the effluent from partial

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nitritation of anaerobic digestion liquor, with the nitrite to ammonium ratio initially fixed at

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its theoretical value of 1.06, and then varied in the range of 1.0-1.375. The performance of

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the MBfR was closely monitored through chemical analysis, and the microbial community

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investigated using Fluorescence in situ hybridization (FISH) and 16S rRNA gene amplicon

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sequencing. The interactions among key organisms, Anammox bacteria, DAMO bacteria and

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DAMO archaea were analysed through mass balance analysis.

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2. Materials and methods

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2.1 MBfR setup

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The MBfR was equipped with 12 bundles of hollow-fibre membrane and was fed

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methane in lumen of membrane through a gas cylinder (Figure 1). Each bundle consisted of

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500 hollow-fibres (non-porous polypropylene hollow-fibre, Teijin Fibres, Ltd, Japan) with a

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length of 30 cm and an inner/outer fibre diameter of 90/200 µm. The total surface area of

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membrane was 1.13 m2. Both ends of the hollow fibres were connected to a feeding gas

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cylinder (95% CH4 and 5% CO2). The gas pressure of interior hollow fibres was manually

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adjusted at 250 kPa by the regulator connected to the gas cylinder and monitored by a gas-

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pressure gauge (Ross Brown, Australia). The feeding gas was forced to penetrate through the

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wall of the hollow fibres, while the non-porous layer allows the creation of a high driving

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force for gas permeation without bubble formation27. 7



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Figure 1. Schematic diagram of the membrane biofilm reactor (MBfR)

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The total volume of the reactor is 2356 mL with a volume of 11 mL inside the hollow

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fibres for gas delivery and a volume of 45 mL for fibres material. As a result, the working

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volume of the MBfR was 2300 mL for liquid flow and biofilm growth. Thus, the specific

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surface area of biofilm is 491 m2/m3. A 20 L feeding tank was used to store synthetic

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wastewater containing nitrite and ammonium. To ensure anaerobic operation of the MBfR, a

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20 L gas bag containing nitrogen gas was connected to the feeding tank. The synthetic

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wastewater was fed to the reactors with a peristaltic pump (BT300-2J, Longerpump, China).

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Complete mixing of the reactor was ensured through liquid recirculation (600 mL/min) using

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a peristaltic pump (Masterflex L/S, USA). A 330 mL overflow bottle was set up to release the

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gas produced in the MBfR and monitor the system pH by a pH meter (Oakton, Australia).

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The system pH was maintained at 7.0-8.0 by manually injecting 1 M HCl or 1 M NaOH

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solutions every day.

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2.2 Operation of MBfR

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The reactor was operated continuously for 627 days, with two phases namely a start-up phase and a continuous phase. During the start-up phase, the MBfR was inoculated with 8


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sludge (400 mL) taken from a parent bioreactor fed with methane, ammonium and nitrate,

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where both Anammox and DAMO microorganisms were enriched in the suspended phase17.

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At the time of inoculation, the volatile suspended solid (VSS) of the parent reactor was

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approximately 3 g/L. Following inoculation, mixed gas (95% CH4 and 5% CO2) was supplied

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to the lumen of the fibres, and the reactor was operated in recirculation mode to establish

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biofilm colonization. To prevent biomass washout through effluent discharge during this

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stage, concentrated nitrite (46 g N/L) and ammonium (46 g N/L) solutions were fed manually

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to maintain the bulk ammonium and nitrite concentrations in the ranges of 0-200 mg N/L and

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0-25 mg N/L.

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From Day 223, the MBfR was turned into continuous operation. The influent

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contained 500 mg NH4+-N/L and 530 mg NO2−-N/L with a ratio of 1:1.06 (Reaction 4).

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The total nitrogen was 1030 mg N/L to mimic effluent from partial nitritation of

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anaerobic digestion liquor. The initial HRT was set at 8 days with a nitrogen loading

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rate at 128.8 mg N/L/d, based on the measured nitrogen removal rate of 120 mg N/L/d

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on Day 223. During the continuous operation, the HRT was stepwise decreased from 8

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days to 1 day when the nitrate was almost completely removed at each condition.

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From Day 588, the ratio of nitrite to ammonium was changed to 1.19 with 470 mg

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NH4+-N and 560 mg NO2--N/L in the influent, with the aim to achieve complete

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nitrogen removal.

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After the reactor reached steady-state, the MBfR nitrogen removal performance at

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different influent nitrite to ammonium ratios (1.0, 1.125, 1.19, 1.25, 1.32 and 1.375)

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was investigated in 6 runs. In all tests, total nitrogen in influent was kept constantly at

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1030 mg N/L. The MBfR was operated continuously, with each test lasting for 4 days.

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During all tests, liquid samples were taken every 12 h from the overflow bottle to determine

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the concentrations of NH4+-N, NO2−-N and NO3−-N. 9



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2.3 Analytical methods Liquid samples of MBfR were taken regularly from overflow bottle to determine the

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concentrations of NH4+-N, NO2−-N and NO3−-N. The concentrations of nitrogenous

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compounds in the samples were measured with a Lachat QuickChem 8000 Flow Injection

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Analyzer (Lachat Instrument, Milwaukee, WI)28. The ammonium and nitrite removal rates

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were determined based on the measured profiles. The total nitrogen removal rate of the

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MBfR was determined by the ammonium removal rate ( RNH + , mg/L/d), the nitrite removal 4

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rate ( RNO− , mg/L/d) and the nitrate accumulation rate ( RNO− , mg/L/d) based on the nitrogen 2

3

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loading rate and effluent quality. Microbial community analysis showed that DAMO archaea,

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Anammox bacteria and DAMO bacteria jointly dominated the microbial community in the

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biofilm (see Section 3.3), suggesting that ammonium oxidation by Anammox (Reaction 1),

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nitrite removal by DAMO bacteria (Reaction 2) and nitrate reduction by DAMO archaea

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(Reaction 3) are the dominating nitrogen conversion reactions. The roles of heterotrophic

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denitrifiers was also evaluated based on the theoretical biomass yields of the Anammox and

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DAMO microorganisms29, biodegradability of yielded biomass27, and the amount of biomass

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washed away with effluent (Supporting Information). The organic matter available for

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denitrification in the MBfR was calculated as only 0.066 g COD/L/d at the final steady stage.

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The contribution of heterotrophic denitrification to the total nitrogen removal rate was

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estimated to be negligible (

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