Phosphorus (P) recovery coupled with increasing influent ammonium facilitated intracellular carbon source storage and simultaneous aerobic phosphorus & nitrogen removal

Accepted Manuscript Phosphorus (P) recovery coupled with increasing influent ammonium facilitated intracellular carbon source storage and simultaneous aerobic phosphorus & nitrogen removal Qing Tian, Linjie Zhuang, Say Kee Ong, Qi Wang, Kangwei Wang, Xuehui Xie, Yanbin Zhu, Fang Li PII:

S0043-1354(17)30142-2

DOI:

10.1016/j.watres.2017.02.050

Reference:

WR 12719

To appear in:

Water Research

Received Date: 21 September 2016 Revised Date:

2 February 2017

Accepted Date: 21 February 2017

Please cite this article as: Tian, Q., Zhuang, L., Ong, S.K., Wang, Q., Wang, K., Xie, X., Zhu, Y., Li, F., Phosphorus (P) recovery coupled with increasing influent ammonium facilitated intracellular carbon source storage and simultaneous aerobic phosphorus & nitrogen removal, Water Research (2017), doi: 10.1016/j.watres.2017.02.050. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Phosphorus (P) Recovery Coupled with Increasing Influent Ammonium

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Facilitated Intracellular Carbon Source Storage and Simultaneous Aerobic

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Phosphorus & Nitrogen Removal

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Qing Tian1*, Linjie Zhuang1, Ong Say Kee2*, Qi Wang1, Kangwei Wang1, Xuehui Xie1, Yanbin

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Zhu1, Fang Li1

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DongHua University, 2999 Shanghai North people’s Road, 201620 P.R. China

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Department of Environmental Science and Engineering

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Department of Civil, Construction, and Environmental Engineering,

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Iowa State University, Iowa 50011 U.S.A.

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E-mail: [email protected]

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12 *Corresponding author

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Qing Tian

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Department of Environmental Science and Engineering

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DongHua University, 2999 Shanghai North people’s Road, 201620 P.R. China

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E-mail: [email protected]

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Abstract

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Under decreasing C/N (from 8.8 to 3.5) conditions, an alternating anaerobic/aerobic

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biofilter (AABF) was used to remove nitrogen and accumulate /recover phosphorus (P)

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from synthetic wastewater.

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additional carbon source (10 L, chemical oxygen demand (COD) =900 mg L-1 NaAC

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solution) in the anaerobic phase to induce the release of P sequestered in a biofilm.

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The increase in PHA storage in the biofilm was characterized with TEM and a

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GC-MS method.

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primarily in the aerobic phase.

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0.238 kg m-3 d-1 at a total empty bed retention time (EBRT) of 4.6 h, the TN removal

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in AABF was reduced from 91.2% to 43.4%, while the P removal or recovery rate

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remained unaffected.

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indicated that the relative abundance of Candidatus Competibacter, Nitrospira and

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Arcobacter increased while the Accumulibacter phosphatis decreased with the

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increase of ammonium loading rate within a short operational period (30 days).

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putative N and P removal pattern via simultaneous nitrification and PHA-based

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denitrification, as well as P accumulation in the biofilm, was proposed and evidenced

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partially.

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process, i.e., simultaneous nitrification and denitrification, P accumulation and carbon

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source regulated recovery can be achieved by the symbiotic functional groups in one

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single biofilm reactor.

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Key words:

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PHA, Intracellular Carbon Source, Phosphorus Recovery, Candidatus Competibacter,

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Simultaneous Phosphorus and Nitrogen Removal

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The AABF was periodically (every 10 days) fed with an

The accumulation of P and removal of total nitrogen occurred

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As the NH4+-N loading rate increased from 0.095 to

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The high-throughput community sequencing analysis

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The research demonstrated that an efficient N removal and P recovery

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1. Introduction

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Emissions of N and P from wastewater can cause eutrophication of water bodies and

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the waste of P resources.

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worldwide issue that must be addressed to solve the problem of the current scarcity of

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P resources (Mbamba et al., 2016; Hukari et al., 2016).

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innovative phosphorus recovery process can convert a traditional biological N and P

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removal (Bio-Nutrient Removal, BNR) process into a novel biological nutrient

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storage and recovery process (Bionutrient Removal-Phosphorus Recovery, BNR-PR).

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Thus, from lab to field studies concerning the retrofitting of an existing BNR process

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have attracted much attention of late (van Loosdrecht & Brdjanovic, 2014; Mehta et

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al., 2015).

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may include: (1) the use of P accumulating organisms (including PAOs and DPAOs)

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to take up P from wastewater under aerobic or anoxic conditions; (2) the application

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of a high amount of organic wastewater (e.g., the filtered supernatant from primary

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sludge hydrolysate, glycerol and biodiesel waste) to induce the anaerobic release of P

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from the biomass to establish high-P-containing solutions; and (3) the recovery of the

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released P from a high-P-containing solution by chemical precipitation or

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crystallization.

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and the recovery of P resources.

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The utilization of different configurations and operational methods using a biofilter to

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sequester, remove and recover P from wastewater has recently been reported (Tian et

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al., 2016; Kodera et al., 2013; Wong et al., 2013).

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biofilm processes is the simultaneous establishment of a high-P-containing solution to

68

allow efficient P recovery by submerging the entire biofilm in a solution that contains

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a high concentration of supplementary organic carbon.

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A strategy for the recovery of P from wastewater is a

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The introduction of an

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The primary characteristics of a newly - developed BNR - PR process

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Such a process provides the benefits of enhanced nutrient removal

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The common feature of these

Consequently, all the of

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biomass in the biofilter can absorb and store the supplied organics as an intracellular

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carbon source (e.g., Poly[3-hydroxybutyrate], P3HB) in the biofilm under anaerobic

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conditions and in a periodic P recovery operation.

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The scarcity of a biodegradable and durable carbon source is a limiting factor for

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efficient, simultaneous N and P removal.

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have employed various strategies for the addition of a supplementary organic carbon

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source to enhance the process of N or P removal.

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media made from a durable organic carbon material (e.g., poly(butylene succinate) or

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poly-epsilon-caprolactone) have been developed and adopted as a slow-release carbon

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source to meet the carbon source demand for the denitrification process used to treat

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wastewater with a low C/N ratio (Ruan et al., 2016; Zhang et al., 2016).

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such practices may not be economical.

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We established a biofilm process based on an alternating anaerobic/aerobic biofilter

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system for P removal and carbon source-induced P recovery (BBPR-CPR) in our

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laboratory (Tian et al., 2016).

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source for P removal during the P accmulation period and only used a small volume

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of a concentrated carbon source (COD=900 mg L-1 and C/N > 40) to extract, enrich

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and recover the P sequestered in the biofilm at the end of the P accumulation cycle.

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The operational mode was different in this respect from the biofilter discussed by

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Wong and Kodera (Kodera et al., 2013; Wong et al., 2013).

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biofilter was only functioned as a post-denitrifying P accumulation/recovery reactor

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and worked with an unchanged supplementary organic load.

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investigation, the biofilter system would play multiple functions - nitrification,

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denitrification, biological P storage - as well as allowing P recovery in a single reactor.

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Furthermore, carbon sources would be used at two different loading rates - the normal

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Recent research and engineering practice

However,

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For example, Novel biofilter

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Research made full use of the wastewater carbon

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In their study, the

However, in our

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loading-rate of the carbon source from wastewater and the higher loading rate of the

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carbon-source supplemented during P recovery.

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biofilm BNR and carbon source-regulated P recovery system (BBNR-CPR system).

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This BBNR-CPR system is assumed to have the following inherent characteristics: (1)

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periodic and alternative exposure to low and high concentrations of organic carbon

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sources, (2) the coexistence of nitrifying, denitrifying, P bio-accumulating, and P

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recovering microbial communities, (3) the potential for the excessive accumulation of

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intracellular carbon by the bacterial community in the biofilm during P recovery.

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view of this, this study reported the effect of P recovery operation on PHA storage in

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the biomass and the effect on N and P removal from a macro- to a micro-scale under

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different N loading conditions.

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NH4+-N to confirm the benefits of P harvesting on N and P removal.

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The goal of the present study include: (1) the characterization of PHA storage and

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accumulation in the biofilm; (2) an investigation of the effect of PHA storage on P and

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N removal under various N loading rates; (3) monitoring of the response of the

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microbial communities to understand the effect of PHA storage on the P and N

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removal pattern.

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introduction of supplementary carbon sources for simultaneous enhancing N removal

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and P recovery in treating the low C/N wastewaters.

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2. Materials and Methods

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2.1 Configuration and experimental design of the reactor

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The experiments were conducted in an up-flow AABF in an air-conditioned chamber

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at a controlled temperature of 25 ± 3 oC.

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Therefore, this study established a

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In

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The influent C/N was decreased by increasing the

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The results of the study will provide a new vision of efficient

As shown in Fig. 1, the system consisted of 5

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a biofilter, an influent tank, an intermediate tank, an effluent tank and a P recovery

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tank. Details about the operational mode, parameters and device in use are listed in

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Table S - 2.

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from the influent tank into the bottom of the biofilter for 3 h and the partially treated

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wastewater was collected (via the P recovery tank) in the intermediate tank.

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end of the anaerobic phase, the biofilter was switched to the aerobic phase, and the

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effluent wastewater that had collected in the intermediate tank was pumped into the

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bottom of the biofilter with aeration.

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biofilter effluent was directed back into the intermediate tank by a time-controlled

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solenoid valve to avoid discharging the phosphate contained effluent.

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remaining 3 h of the aerobic phase, the biofilter effluent was collected in the effluent

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tank (see Fig. 1).

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d-1 in the anaerobic phase and 0.44 m3 m-2 d-1 in the aerobic phase with an empty bed

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retention time (EBRT) of 2.3 h.

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compressor and solenoid valves was controlled by a programmable logic controller.

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Air was supplied to the biofilter at a flow rate of 0.25 m3 h-1.

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concentration at the bottom and the top section of the AABF in the aerobic phase was

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maintained at or above 2.0 and 3.0 mg L-1, respectively.

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were the influent TN load (originating from the influent NH4+-N and NO3--N) and the

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ratio of the total carbon added to the total nitrogen added.

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performed during the entire operation.

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The biofilter was inoculated with activated sludge obtained from a local municipal

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wastewater treatment plant employing an A/A/O process.

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AABF reached a steady-state after the AABF was inoculated and had acclimated for

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approximately 40 days.

In the anaerobic phase in the biofilter, synthetic wastewater was pumped

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At the

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For the first 2 h of the aerobic phase, the

For the

The average hydraulic loading rate of the biofilter was 0.44 m3 m-2

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The operation of the AABF diaphragm pumps, air

The dissolved oxygen

The variable parameters

No back washing was

The operation of the

The steady-state operation lasted for 30 days and the feed 6

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NH4+-N remained at approximately 24 mg L-1 during that period.

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NH4+-N was deliberately raised from 0.095 kg m-3 d-1 in run I to 0.143, 0.189 and

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0.238 kg m-3 d-1 in runs II, III and IV, respectively (day 86 to day 115, as shown in

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Table 1), in order to accelerate growth and accumulation of nitrifiers .

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was operated under an average COD and P load of 0.63 kg m-3 d-1 and 0.059 kg m-3

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d-1, respectively, and P was harvested every 10 days.

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AABF with small volumes (V=10 L) of concentrated sodium acetate solution (COD

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of 900 mg L-1) under anaerobic conditions to induce the release of the P from the

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biofilm.

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This operational mode was designated as the carbon source-regulated P recovery

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(CPR) mode and was designed to remove or effectively harvest the P in the biofilm of

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the AABF.

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NH4+-N was decreased to 36 mg L-1 and 174 mg L-1 KNO3 was added to the influent

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to keep the TN load at 0.238 kg m-3 d-1 to observe effect of NO3- on PHA consumption

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in the anaerobic phase.

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2.2 Sampling program and analytical methods

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All the water samples in this study were collected from an effluent sampling port at

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the top of the biofilter at 1 h intervals during the anaerobic/aerobic phases.

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distance between the effluent water sampling port and the bottom of AABF was 1.0 m.

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The water level in the biofilter was maintained at 0.05 m higher than the top surface

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of the quartz sand in the biofilter.

The influent

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The AABF

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P was harvested by feeding the

The resulting high P effluent was collected in the P recovery tank (Fig. 1).

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In the final operational period (run V, days 116-125), the influent

The

COD, TP and soluble P, NH4+-N, TN, NO2--N, 7

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NO3--N and total suspended solid (TSS) were measured using Standard Methods

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(APHA, AWWA, WPCF, 1999).

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monitored using an ORP/pH/conductivity meter (WTW pH 3310, Munich, Germany).

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Biofilm samples were obtained at the end of the anaerobic phase of each operational

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stage (e.g., P accumulation and P recovery) in different runs before changing the

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operational conditions.

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community population were obtained from the S1 biomass sampling port, which was

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50 cm above the bottom of the biofilter.

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in the biofilter, around 40 g wet sand particles were taken from the biofilter each time

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to make sure of obtaining the representative biofilm samples.

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were divided into three 10 ml sterilized centrifugation tubes, one for PHA analysis,

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one for DAN extraction and the last one for sample reservation.

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attached with biofilm were put into the 100 ml flask with 50 ml sterilized saline to

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detach the biofilm by rotating the flask for 5 min.

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concentrated by centrifugation at 8,000 rpm for 10 min at 4 °C and stored at −80 °C

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for molecular analysis.

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2.3 Analysis of intracellular carbon source

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The biofilm samples were stained with a DAPI solution (40 ng·µL-1) for 60 min in

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darkness and observed under a fluorescence microscope at emission wavelengths of

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537 – 591 nm for the biofilm containing poly-Ps (Serafim et al., 2002)

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(Sigma-Aldrich, Schnelldorf, Germany).

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of Nile Red solution (200 ng·µL-1) and dried on a flame for a few seconds and was

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Water temperature, conductivity and pH were

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The biomass samples used to study the biofilm bacterial

The sand particles

The sand particles

The biofilm suspension was then

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For the uneven distribution of the biofilm

The biofilm was also stained with a drop

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observed the intracellularly stored PHBs under a florescent microscope (Nikon

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ECLIPSE 80i; Tokyo, Japan).

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sand were rinsed 5 times with a NaCl solution (8g·L-1) and fixed in 2.5%

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glutaraldehyde for 2 h at 4 °C.

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series for 20 min and thoroughly dried in a vacuum freeze dryer for 24 h.

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PHA and poly-P granules inside the cells were examined with a TEM (Hitachi

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HT7700, Tokyo, Japan) at an acceleration voltage of 120 kV.

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staining of the cells included glutaraldehyde fixation, Pb (NO3)2 staining,

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ethanol-acetone dehydration and epoxy resin embedment (Gunther et al., 2009).

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The PHAs were quantified using the method proposed by Tan with a few

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modifications (Tan et al., 2014).

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mg) were suspended in 2 ml of methanol containing 10% (v/v) H2SO4, and incubated

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at 105°C for 6 h.

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monomers was extracted by chloroform and analyzed using a model Shimadzu

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QP2010 Plus gas chromatograph-mass spectrometer (QP - 2010, Shimadzu, Japan)

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with a DD-5 column (30 m length × 0.25 mm diameter × 0.25 µm thickness,

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Shimadzu, Japan).

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characteristic peaks and searching and comparing the mass spectra against standard

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references in the library of National Institute of Standards and Technology database

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(NIST11, Gaithersburg, MD, U.S.).

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2.5 Statistical analysis

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A statistical analysis of the experimental data was conducted using IBM SPSS

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Statistics (21.0) software (http://www-01.ibm.com/software/analytics/spss/).

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one-way ANOVA (analysis of variance) was used to test whether a certain factor

In addition, the detached biofilm samples from the

The samples were then dehydrated with an ethanol

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Then, the

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Pretreatment and

In brief, lyophilized, weighed biomass samples (30

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The organic phase containing the resulting methyl esters of

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PHAs composition was determined by an analysis of the

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impacted an observed variable.

Pearson’s correlation coefficient was applied to

211

quantify the relationship between two parameters.

212

indicated statistically significance.

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2.6 Microbial community analysis

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2.6.1

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All the biofilm samples were sent to Majorbio Bio-Pharm Technology Co., Ltd

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(Shanghai, China) for high-throughput sequencing on an Illumina MiSeq platform

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(Illumina, San Diego, U.S.).

218

E.Z.N.A.® Soil DNA kit (Omega Bio-Tek, Inc., Norcross, GA, U.S.) and the quality

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of the extraction was assessed using a 2% (w/v) agarose gel electrophoresis (Ma et al.,

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2015).

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by PCR (ABI GeneAmp® 9700, CA, U.S.) and the barcode was an eight-base

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sequence unique to each sample.

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followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and a final

224

extension

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GTGCCAGCMGCCGCGG-3’) and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’).

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The PCR reactions were performed in triplicate in a 20 µL mixture containing 4 µL of

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5 × FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of

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FastPfu Polymerase (TransGen, Shanghai, China), and 10 ng of template DNA.

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2.6.2

230

Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA

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Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the

232

manufacturer’s instructions and quantified using the QuantiFluor™ -ST (Promega, WI,

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U.S.).

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An α = 0.05 and P < 0.05

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DNA extraction and PCR amplification

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The biofilm DNA extraction were carried out using the

The V4-V5 regions of the bacterial 16S ribosomal RNA gene were amplified

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°C

for

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min

using

the

primers

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(5’-barcode-

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The detailed PCR program was 95 °C for 2 min,

Illumina MiSeq sequencing

Purified amplicons were pooled in equimolar amounts and paired-end 10

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sequenced (2 × 250) on an Illumina MiSeq platform

according to standard

235

protocols.

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2.6.3

237

Raw fastq files were demultiplexed, and quality-filtered using QIIME (version 1.9.1)

238

with the following criteria: (i) The 250 bp reads were truncated at any site with an

239

average quality score <20 over a 50 bp sliding window, discarding truncated reads

240

shorter than 50bp. (ii) exact barcode matching, a 2 nucleotide mismatch in primer

241

matching, and reads containing ambiguous characters were removed. (iii) only

242

sequences with an overlap greater than 10 bp were assembled according to the

243

overlapping sequences.

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GenBank with an accession number SRP090073.

245

assembled were discarded.

246

using a 97% similarity cutoff via Usearch (version 7.1 http://drive5.com/uparse/) and

247

chimeric sequences were identified and removed using UCHIME (version 7.1).

248

taxonomy of each 16S rRNA gene sequence was analyzed by the RDP Classifier

249

(http://rdp.cme.msu.edu/) against the Silva (SSU123)16S rRNA database using a

250

confidence threshold of 70% (Tan et al., 2014).

251

2.6.4

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Community diversity index (Chao, Ace, Simpson, Coverage, Shannon diversity index)

253

and rarefaction curves were generated using the MOTHUR program. A heatmap

254

analysis of the 100 most abundant genera in each group and rank-abundance were

255

both conducted using the R software (https://www.r-project.org/).

256

2.6.5

257

Fisher’s exact test was used to calculate and compare the significance of the

258

differences between the populations of the two samples and to identify the species

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Post-processing of sequencing data

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All the sequences have been deposited in the NCBI Reads which could not be

Operational Taxonomic Units (OTUs) were clustered

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The

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Species richness and diversity determination

Analysis of differences between sample populations

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with the most difference in their relative abundance using the STAMP software

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(http://kiwi.cs.dal.ca/Software/STAMP)

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3. Results and Discussion

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3.1 Intracellular carbon source storage

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The steady-state operation lasted for 30 days, and the average concentration of the

265

influent NH4+-N remained at 24 mg L-1 during that period.

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wastewater was converted into poly-P by the PAOs in the biomass and established a

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pool for poly P in the biofilm of the AABF, as shown in Fig. 2 (a), an image of the

268

biofilm stained with DAPI.

269

the biomass that was characterized by a blue florescence from the DNAs of the active

270

cells.

271

cells of the PAOs, as shown in Figs. 2 (c) and (e), which are TEM photographs of the

272

biomass.

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supplements of carbon source were fed to the AABF during an anaerobic phase.

274

optimize the amount of the supplementary carbon source, the effect of carbon source

275

concentration on the PHA content in the biomass was investigated using a 400, 900

276

and 2000 mg L-1 NaAC solution in batch tests with the biomass obtained from run I.

277

A COD=900 mg L-1 NaAC solution (10 L) was selected as the best supplementary

278

carbon source and was fed to the AABF in the anaerobic phase at the end of every

279

P-accumulation cycle.

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The soluble P in the

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A strong yellow or green florescence was emitted from

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The poly P granules resembled dense black granules of various types in the

To harvest the biomass-sequestered P, periodic (every 10 days)

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To

This operation resulted in a high P-containing solution (the 12

ACCEPTED MANUSCRIPT maximum 236 mg L-1) emitted from the AABF and the high absorption and storage of

281

the carbon source from the P recovery solution to the biomass in the AABF.

282

The first evidence of the formation and storage of the PHA clusters in the biofilm was

283

the strong red florescent signals from the Nile-red stained biomass (Fig. 2 (b)), and

284

the second was the white granules in the TEM photograph of the biomass (Fig. 2 (d)

285

and (f)).

286

end of run IV (Fig. 2 (f)) were much greater than the PHA granules at the end of run I

287

(Fig. 2 (d)), which suggested a higher PHA accumulation in the biomass in the

288

different AABF runs.

289

by GC-MS.

290

from 9.84% – 13.86% (before P recovery) to 20.14% – 36.80% after the P recovery,

291

when the average AABF influent ammonium concentration was raised from 24 mg L-1

292

to 60 mg L-1, respectively.

293

3.2 Simultaneous aerobic phosphorus and nitrogen removal

294

The TN in the anaerobic influent and effluent or the aerobic influent all contained

295

NH4+-N in run I – run VI, and no NO2- and NO3- were present in the biofilter influent

296

(the simulated wastewater).

297

decreased in the aerobic phase and a certain amount of NO3- was also detectable in the

298

effluent.

299

growth of the biofilm on the packing of the AABF and extensive agglomeration of the

300

biomass in localized sections of the biofilter were observed from the transparent

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The size and amount of the white PHA granules in the biomass from the

Thirdly, the PHA accumulation in the biomass was quantified

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Fig. 3 shows that the percentage of the stored PHAs in the biomass rose

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Thus, PHA storage and P harvesting were simultaneous.

Fig. 4 shows that both the effluent TN and the NH4+-N

Although the DO of the biofilter reached a high of 3 – 4 mg L-1, the uneven

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plexiglass of AABF.

This biomass agglomeration could prevent the efficient

302

penetration of dissolved oxygen deeper into the biofilm and the low DO level led to

303

denitrification.

304

amount of the influent NH4+-N was removed via biomass assimilation. Most of the

305

influent TN removed in the aerobic phase was removed by a simultaneous aerobic

306

nitrification and denitrification (SND) pathway in the AABF.

307

The denitrification efficiency was greatly dependent on the availability of an

308

extracellular or intracellular carbon source.

309

extracellular carbon source) is an important pre-requirement for SND.

310

bulk COD in the aerobic phase of the AABF was kept below 40 mg L-1.

311

amount of carbon could only remove a theoretical maximum of 9 mg L-1 of TN.

312

More than 25 mg L-1 of TN were removed during the aerobic phase of the AABF,

313

which demostrated that the SND driven by intracellular carbon source occurred and

314

accounted for the aerobic nitrogen removal during the operation of the AABF.

315

The influent NH4+-N was thoroughly depleted, and the average TN removal was as

316

high as 91.2%, when the average influent NH4+-N concentration was 24 mg L-1, as

317

shown in Fig. 4 (a).

318

phase in the AABF peaked at 89.1%.

319

remained at 69.5%,

320

from 36 to 48 to 60 mg L-1.

321

to 60 mg L-1

322

have occurred because the alkalinity was not adjusted back to the original value (the

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Since little excess biomass was generated from the AABF, a limited

However, the Such an

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A high level of bulk COD (i.e., an

Furthermore, the average removal of influent TN in the aerobic The aerobic TN removal still respectively

52.8%, and 39.4% when the influent NH4+-N was increased An increase of the average influent NH4+-N from 48

impacted the NH4+-N removal, which decreased by 8%.

14

This could

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effluent pH decreased to 6.6 ), which prevented complete nitrification when the

324

influent NH4+-N was increased (7.14 mg CaCO3 alkalinity was needed to oxidize

325

completely 1 mg of influent NH4+-N).

326

decreased by 13.4% (from 52.8% to 39.4%), which was 5% higher than the average

327

decrease of NH4+-N removal.

328

low influent alkalinity and low PHAs storage with an increased NH4+-N loading rate.

329

However, an increase of the influent NH4+-N from 24 to 48 mg L-1 did not have a

330

significant effect on the P aerobic uptake rate (analysed by a one-way ANOVA,

331

P=0.115).

332

conditions, for example, the average TP removal was 90.6%, 92.6% and 89.3% at

333

average influent NH4+-N rates of 24, 36 and 48 mg L-1, respectively.

334

amount of influent NH4+-N in a conventional BNR system (e.g., A/A/O or SBR

335

system) was reported to result in a greater amount of NO3- remaining in the bioreator,

336

which would suppress the subsequent anaerobic P release.

337

effluent to the anaerobic phase occurred in this biofilter system in the subsequent

338

anaerobic/aerobic cycle; only 21.5% of effluent NO3- remained in the biofilter (78.5%

339

of the NO3- in the effluent was emitted directly, as calculated according to the mass

340

balance of the treated water).

341

the carbon source was stored as PHAs in the biomass, as shown in Fig. 3.

342

PHA storage provided sufficient energy for P uptake and a carbon source for SND in

343

the aerobic phase.

344

phase of the AABF, the effluent NO3- was less than 10 mg L-1, so the effluent that

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In addition, the average aerobic TN removal

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The decreased TN removal probably resulted from

A higher

No recycling of aerobic

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The AABF had a high TP removal capacity under different NH4+-N

In addition, during P havesting, a high percentage of This high

Furthermore, because of the occurance of SND during the aerobic

15

ACCEPTED MANUSCRIPT 345

remained in the biofilter did not have a significant effect on anaerobic release of TP or

346

its removal in the next cycle.

347

during the aerobic phase and an increase of the influent NH4+-N did not impact P

348

uptake in the aerobic phase of AABF.

349

3.3 Dynamic change of bio-communities with increased NH4+-N loads

350

To extend the understanding of the periodical carbon source supplement effect to

351

BNR efficiency, the composition of the microbial community in the biofilm samples

352

were analyzed with high-throughput methods (Illumina MiSeq sequencing).

353

Amplicons of bacterial 16s rRNA genes were sequenced and assigned to phylotypes.

354

The results of the rarefaction analysis (Fig. S1) indicated that sufficient sequence

355

coverage (average reads of 21519 per sample) were obtained for each sample.

356

Sequences of the three biofilm samples were clustered and obtained 617, 565 and 551 OTUs,

357

respectively. These OTUs belongs to 290, 277 and 261 genera for the biofilm samples

358

obtained at the end of Run I, Run II and Run IV, respectively.

359

community species composition and structural changes with AABF operational

360

conditions, the Ace, Shannon and Simspon diversity indexes were determined to

361

reflect the individual number, community species and the uniformity of the

362

distribution.

363

the influent NH4+-N increased, indicating a decrease in the total number of species in

364

the biofilm.

365

the alpha diversity of a microbial community in a sample (Edward Hugh Simpson,

To demonstrate the

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In conclusion, a high removal rate of TN occurred

As shown in Table S4, and the Ace index decreased from 680 to 632 as

In addition, the Simpson and Shannon indexes are often used to reflect

16

ACCEPTED MANUSCRIPT 366

1949).

A higher Simpson index or a lower Shannon value indicates a lower

367

microorganism community diversity.

368

ammonia NH4+-N from 24 to 36 and 60 mg L-1 the decreased the diversity of the

369

biofilm microorganism community (the Shannon index dropped from 4.87 to 4.66,

370

4.63 and the Simpson index increased from 0.0174 to 0.0197, 0.0198, respectively).

371

The relative abundance of the top 15 genera in the biofilm samples from the different

372

operational runs are shown in Fig. 5.

373

of OTUs assigned to a genus divided by the total OUT numbers of a biofilm sample.

374

A Fisher’ exact test bar plot was provided to differentiate the bacterial genera which

375

relative abundance increased or decreased significantly (P<0.05) in the AABF

376

biofilms, as shown in Fig. S1.

377

Competibacter), Lewinella, Nitrospira, Thiodictyon, Rhodobacter, Acinetobacter,

378

Zoogloea, Hydrogenophaga were distinguished by the difference of magnitude of

379

their relative abundance in the runs of Run I and Run IV.

380

phenomenon in the community composition was the fraction of Ca. Competibacter,

381

which increased from 12.89% to 20.12% as the influent ammonium concentration

382

increased from 24 to 60 mg L-1 within 30 days.

383

been reported to produce and accumulate PHAs under anaerobic conditions, but the

384

cells accumulate glycogen instead of polyphosphate under aerobic conditions.

385

they were called glycogen accumulating organisms (GAOs) or Ca. Competibacter of

386

competition for carbon source with PAOs under anaerobic conditions in BNR systems.

387

However, some subgroups of GAOs were reported to be capable of reducing nitrate to

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Table S4 shows that an increase of influent

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The relative abundance was defined as the sum

The most notable

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The genus of Candidatus Competibacter (Ca.

17

Members of Ca. Competibacter has

Thus

ACCEPTED MANUSCRIPT 388

nitrite, and or to nitrogen using PHAs as an intracellular carbon source (Coats et al.,

389

2011a; McIlroy et al., 2014).

390

samples were further analyzed using the clustering method of phylogenic tree (Fig. S3)

391

(McIlroy et al., 2014; Mao et al., 2016).

392

members of Ca. Competibacter (e.g. members belonging to Clade I) can denitrify

393

(Bassin et al., 2012).

394

present in the three biofilm samples, and their relative abundance remained high.

395

The relative abundance of Ca. Competibacter varied with an increase in the influent

396

NH4+-N, but it did not significantly impact P uptake by the PAOs.

397

species were reported as potential denitrifers that have the capacity to produce PHA,

398

e.g., members of genera Rhodobacter (Arumugam et al., 2014; Granger et al., 2008)

399

and Hydrogenophaga (Hwang et al., 2006; Reddy et al., 2016), which also presented

400

with an increase in the influent NH4+-N.

401

relative abundance of the microbial communities was for the genus Nitrospira.

402

members of Nitrospira was considered as chemolithoautotrophic nitrite oxidizers

403

(NOB), and it was recently reported that a member of the genus Nitrospira

404

(Candidatus Nitrospira defluvii) can function as both an ammonia oxidizer (AOB)

405

and NOB (Daims et al., 2015).

406

The enrichment of PHA-producing bacteria and poly-P accumulating bacteria (PAOs)

407

can be achieved by cyclic P accumulation and harvesting (which induced a high-C

408

loading rate and reduced the polyphosphates content of the biofilm).

409

enriched Pseudomonas (members of Pseudomonadaceae of γ-Proteobacteria) with a

The subgroups of Ca. Competibacter in the biofilm

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In addition, some denitrifying bacteria, e.g.,

In addition, other

The other population with the increased Most

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The putative denitrifying Ca. Competibacter (clade I) were

18

For example,

ACCEPTED MANUSCRIPT 410

98% relative abundance were observed in our previous study (Tian et.al., 2016).

The

411

P bioaccumulation by the members relating Pseudomonas of γ-proteobacteria had

412

been reported early by Sresb (Sresb, et al. 1985) and confirmed by many researchers,

413

e.g. C.K. Lin (Lin et al. 2003).

414

Accumulibacter phosphatis belonging to Clade II CD as indicated by Fig. S4, also the

415

putative denitrifying PAO, were presented in the three biofilm samples (He et al.,

416

2007; Kim et al., 2013; Welles et al., 2016).

417

and 2.99% in the end of run I, run II and run IV, respectively.

418

3.2, the P removal was unaffected.

419

production and increased storage in the biofilm during P harvesting (when the AABF

420

was supplied with high amounts of carbon) as shown in Fig. 3.

421

In summary, symbiotic functional groups including nitrifiers, denitrifiers (including

422

the DPAOs and DGAOs) were present in all there biofilm samples.

423

DPAO (e.g belonging to PAO Clade II ) and DGAO (e.g belonging to GAO Clade I)

424

and nitrifiers present in the biofilm and perfoming aerobic simutaneous nitrification

425

and denitrification with the increase of NH4+-N loading.

426

habitating in the aerobic zone or biofilm layers using PHA as an energy source to take

427

up P from the bulk solution, as shown in Fig. 6.

428

zone in the biofilm could use PHA as an electron donor and to take up P using NO3- or

429

the NO2- as electron acceptors for dentrification.

430

NO3- reduction by the DGAOs.

431

occurred via the SND pathway and was a result of the combination of the

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In this study, members of Candidatus

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Its relative abundance were 6.26%, 3.68%

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As indicated in Sec.

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This result explained the effective PHA

Since both the

It is possible that the PAOs

While the DPAOs in the anoxic

The NO2- might be produced from

Therefore, the effective TN removal probably

19

ACCEPTED MANUSCRIPT accumulation and storage of P and PHAs in the biofilm.

The appreciable presence of

433

nitrifying and denitrifying micro-communities probably was the result of the adaption

434

of microbial communities to the increasing NH4+-N.

435

3.4 Implications and recommendations

436

The quantity and quality of the available carbon source are the key to successful BNR

437

processes.

438

study.

439

pilot-scale wastewater treatment engineering (assuming the treatment capacity of the

440

treatment capacity of 1 m-3·d-1), the cost of using sodium acetate as the supplementary

441

carbon source would be 0.012

442

Other choices for cheap supplementary carbon source can be glycerol or methanol.

443

Their calculated cost as the supplementary carbon source are 0.011

444

glycerol (price of 0.43

445

(price of 0.34

446

presented in Table S - 2 and Table S - 3.

447

considered in the future study if applying the system for pratical application.

448

There are more advantages to carry out the performance except for the benefits from P

449

recycling.

450

carbon source (i.e., the supplementary carbon source and the wastewater carbon

451

source) with different characteristics.

452

produced PHA is associated with the characteristics of substrates (concentration,

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432

kg-1) was applied in a practical

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Given the NaAC (58% purity, price of 0.19

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Sodium acetate was used as the supplementary carbon source in this

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m-3· d-1, which appeared to be economically feasible.

kg-1 at 99% purity) and 0.007

m-3 d-1 for methanol

The detailed calculation information is More cheaper carbon source could be

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kg-1 at 99% purity).

m-3· d-1 for

First, the biofilm in the biofilter is necessarily subjected to two types of

It had reported that the quantity of microbial

20

ACCEPTED MANUSCRIPT 453

salinity and alkalinity of carbon) (Tan et al., 2009).

454

supplementary carbon source in this investigation may not the best choice for

455

supplementary carbon source.

456

production and the fermented sludge had been reported as effective alternative carbon

457

source for denitrification (Bodik et al., 2009, Ji & Chen, 2010).

458

hydrolyzation process of biodiesel wastewater, a great deal of propionic acid would be

459

produced and would be a promising supplementry carbon source.

460

hydrolysate of biodiesel as the supplementray carbon source to the system, the carbon

461

from the biodiesel waste could be effectively recycled in this way.

462

investigation is still needed to optimize the types and quantity of supplementary and

463

to improve the efficiency of BNR.

464

All in all, in view of the special characteristics of “alternating exposure to the carbon

465

source from the wastewater and the supplementary” and “symbiosis of different

466

functional microorgamisum groups” for the biofilter system in our investigation, we

467

believe that the novel biofilm BNR-CPR bring about a new BPR configuration and

468

operation strategy for the recovery of P (and carbon source) from wastewaters.

469

step futher, it also provids an novel, simple and economical pattern to enhace TN

470

removal from the low C/N wastewaters.

471

4. Conclusions

472

1.

473

Using acetate as the

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For example, the wastewater from the biodiesel

If we apply the

Further

One

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Furthermore, in the

Periodic supplementation with a high concentration of carbon to an AABF to induce a high release of biomass-sequestered P and an increased storage of PHAs 21

ACCEPTED MANUSCRIPT 474

in microorganisms compared to the PHAs storage before P harvesting in the

475

AABF biofilm.

476

2.

The influent NH4+-N was removed directly from the AABF in the aerobic phase via PHA-based simultaneous nitrification and denitrification in the aerobic phase

478

of the AABF.

479

3.

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477

The presence of Ca. Competibacter (member of Ca. Competibacter Clade I) with the production of PHA and a nitrate and nitrite reduction capacity and the

481

members of Nitrospira were observed to accompany an increase of the influent

482

NH4+-N. 4.

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480

Because the spontaneous storage of PHAs occurred during P harvesting, the adverse impact of the high GAO growth rate on P removal was avoided.

485

Increasing the influent NH4+-N did not significantly impact TP removal. The

486

removal of influent NH4+-N benefited from the increased PHA storage during the

487

carbon source-regulated P recovery.

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488

Acknowledgments

490

The authors acknowledge the editor and anonymous reviewers’ suggestion to improve

491

the paper quality and the financial support from the Natural Science Foundation of

492

Shanghai (16ZR1402000) and the National Natural Science Foundation of China

493

(51478099).

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494

22

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Daims, H., Lebedeva, E.V., Pjevac, P., Han, P., Herbold, C., Albertsen, M., Jehmlich, N., Palatinszky, M., Vierheilig, J., Bulaev, A., Kirkegaard, R.H., von Bergen, M., Rattei, T., Bendinger, B., Nielsen, P.H., Wagner, M. 2015. Complete nitrification by Nitrospira bacteria. Nature, 528(7583), 504-516.

Granger, J., Sigman, D.M., Lehmann, M.F., Tortell, P.D. 2008. Nitrogen and oxygen isotope fractionation during dissimilatory nitrate reduction by denitrifying bacteria. Limnology and Oceanography, 53(6), 2533-2545.

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Gunther, S., Trutnau, M., Kleinsteuber, S., Hause, G., Bley, T., Roske, I., Harms, H., Muller, S. 2009. Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling. Applled Environmental Microbiology, 75(7), 2111-2121. He, S., Gall, D.L., McMahon, K.D. 2007. "Candidatus Accumulibacter" population structure in

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Ji, Z., Chen, Y. 2010. Using sludge fermentation liquid to improve wastewater short-cut nitrification-denitrification and denitrifying phosphorus removal via nitrite. Environ Sci Technol, 44(23), 8957-63. Kim, J.M., Lee, H.J., Lee, D.S., Jeon, C.O. 2013. Characterization of the Denitrification-Associated Phosphorus Uptake Properties of "Candidatus Accumulibacter phosphatis" Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal. Applied and Environmental Microbiology, 79(6), 1969-1979. Kodera, H., Hatamoto, M., Abe, K., Kindaichi, T., Ozaki, N., Ohashi, A. 2013. Phosphate recovery as 23

ACCEPTED MANUSCRIPT concentrated solution from treated wastewater by a PAO-enriched biofilm reactor. Water Research, 47(6), 2025-2032. Lin, C.K., Katayama, Y., Hosomi, M., Murakami, A. and Okada, M. 2003. The characteristics of the bacterial community structure and population dynamics for phosphorus removal in SBR activated sludge processes. Water Research, 37(12), 2944-2952. Ma, J.X., Wang, Z.W., He, D., Li, Y.X., Wu, Z.C. 2015. Long-term investigation of a novel electrochemical membrane bioreactor for low-strength municipal wastewater treatment. Water

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Research, 78, 98-110.Mbamba, C.K., Flores-Alsina, X., Batstone, D.J., Tait, S. 2016. Validation of a plant-wide phosphorus modelling approach with minerals precipitation in a full-scale WWTP. Water Research, 100, 169-183.

Mao, Y.P., Wang, Z.P., Li, L.G., Jiang, X.T., Zhang, X.X., Ren, H.Q., Zhang, T. 2016. Exploring the Shift in Structure and Function of Microbial Communities Performing Biological Phosphorus

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McIlroy, S.J., Albertsen, M., Andresen, E.K., Saunders, A.M., Kristiansen, R., Stokholm-Bjerregaard, M., Nielsen, K.L., Nielsen, P.H. 2014. 'Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity. Isme Journal, 8(3), 613-624.

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Mehta, C.M., Khunjar, W.O., Nguyen, V., Tait, S., Batstone, D.J. 2015. Technologies to Recover Nutrients from Waste Streams: A Critical Review. Critical Reviews in Environmental Science and Technology, 45(4), 385-427.

Reddy, M.V., Mawatari, Y., Yajima, Y., Satoh, K., Mohan, S.V., Chang, Y.C. 2016. Production of poly-3-hydroxybutyrate

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poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

P(3HB-co-3HV) from synthetic wastewater using Hydrogenophaga palleronii. Bioresource Technology, 215, 155-162.

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Ruan, Y.J., Deng, Y.L., Guo, X.S., Timmons, M.B., Lu, H.F., Han, Z.Y., Ye, Z.Y., Shi, M.M., Zhu, S.M. 2016. Simultaneous ammonia and nitrate removal in an airlift reactor using poly(butylene succinate) as carbon source and biofilm carrier. Bioresource Technology, 216, 1004-1013. Serafim, L.S., Lemos, P.C., Levantesi, C., Tandoi, V., Santos, H., Reis, M.A.M. 2002. Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating

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J.-Y. 2014. Start a Research on Biopolymer Polyhydroxyalkanoate (PHA): A Review. Polymers, 6(3), 706-754.

Tan, G.Y.A., Chen, C.L., Ge, L.Y., Li, L., Wang, L., Zhao, L., Mo, Y., Tan, S.N., Wang, J.Y. 2014. Enhanced

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Tian, Q., Ong, S.K., Xie, X., Li, F., Zhu, Y., Wang, F.R., Yang, B. 2016. Enhanced phosphorus recovery and biofilm microbial community changes in an alternating anaerobic/aerobic biofilter. Chemosphere, 144, 1797-1806. Van Loosdrecht, M.C.M., Brdjanovic, D. 2014. Anticipating the next century of wastewater treatment. Science, 344(6191), 1452-1453. Welles, L., Lopez-Vazquez, C.M., Hooijmans, C.M., van Loosdrecht, M.C.M., Brdjanovic, D. 2016. Prevalence of 'Candidatus Accumulibacter phosphatis' type II under phosphate limiting 24

ACCEPTED MANUSCRIPT conditions. Amb Express, 6, 12. Wong, P.Y., Cheng, K.Y., Kaksonen, A.H., Sutton, D.C., Ginige, M.P. 2013. A novel post denitrification configuration for phosphorus recovery using polyphosphate accumulating organisms. Water Research, 47(17), 6488-6495. Zhang, Q., Ji, F.Y., Xu, X.Y. 2016. Effects of physicochemical properties of poly-epsilon-caprolactone on nitrate removal efficiency during solid-phase denitrification. Chemical Engineering

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Journal, 283, 604-613.

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Table – 1 Experimental design and the parameter changes Supplementary Synthetic wastewater

Total

carbon source

C/N

solution

Run -1

Ⅰ (day 40 – 85)

b

Ⅱ (day 86 – 95)

b

Ⅲ (day 96 – 105)

b

Proposed

NO3--N

TN

-1

COD

C/N

mg L

mg L

22.41±1.85

0

24

7.5

35.12±1.59

0

36

5

mg L

47.44±1.41

0

48

3.75

Ⅳ (day 106 – 115)

b

57.65±1.42

0

60

3

Ⅴ (day 116 – 125)

c

34.24±0.62

23.86±0.84

60

3

C/N

mg L-1

-1

900

8.8

900

5.9

900

4.4

900

3.5

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NH4+-N

900

-1

3.5

-1

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a, Influent COD and TP were kept unchanged (average COD=186.3 mg L , TP=15.9 mg L ) through all the experimental time b, Influent TN load only originated from NH4+-N c, Influent TN load originated from the combination of NH4+-N and NO3- - N

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2 3 4 5 6 7 8

a

1

ACCEPTED MANUSCRIPT Table S – 1 The detailed operation unit process parameters and devices in use

Wastewater treatment were treated & P sequestered as poly-P in the biofilm

Operation Alternating anaerobic/aerobic Influent was pumped from influent tank to the biofilter

Duration 8h 3h

2h

Aerobic

3h

Carbon source induced P releasing & PHA storage

Anaerobic

5h

P recovery by struvite crystallization

Not discussed in this study

(1) Influent pump (3) Biofilter (8) Influent tank

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(2) Intermediate pump (3) Biofilter (5) Air compressor (7) Intermediate tank (4) Effluent valve (3) Biofilter (5) Air compressor (7) Intermediate tank

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Aerobic

Device in use

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Function

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(1) Influent pump (3) Biofilter (6) P recovery tank

(6) P recovery tank

ACCEPTED MANUSCRIPT

Carbon source*

Price € (RMB) kg-1

Theoretical dosage g m-3

Methanol Sodium acetate Glycerol

0.34(2.5) 0.19(1.4) 0.43(3.2)

0.667 2.210 0.834

Budget Supplementary carbon source -3 € (RMB) m

Wastewater treated** € (RMB) m-3

0.23(1.67) 0.42(3.10) 0.36(2.67)

0.007(0.05) 0.012(0.09) 0.011(0.08)

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15 16 17 18 19

Table S – 2 The theoretical budget for the three types of supplementary carbon source

Note: *, The detailed characteristic parameters of the supplementary carbon source is listed in Table S – 3 **, Calculated on the basis of the supplementary carbon source dosage rate, VSCS: VW = 0.03

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12 13 14

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ACCEPTED MANUSCRIPT Table S – 3 The characteristics of three types of supplementary carbon sources supplementary carbon source (industry grade)

Physical state

Density g cm-3

CAS NO.

Purity (%)

Information Source

Methanol

liquid

0.792

67-56-1

>99

Sodium acetate

solid

222

6131-90-4

58

Glycerol

liquid

1.27

56-81-5

>99

https://detail.1688.com/of fer/45070334320.html. https://detail.1688.com/of fer/1147563439.html?trac elog=p4p, https://detail.1688.com/of fer/535899269801.html?t racelog=p4p

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4

ACCEPTED MANUSCRIPT Table S - 4 Community diversity indexes in different biofilm samples

Sample ID

0.97 Reads

OTU

ace

coverage

680 Run I

21519

617

0.995

(660,711) 659

Run II

21519

565

0.995

(630,700) 632

21519

551

0.995

(606,669)

Simpson index 0.0174

(4.85,4.89)

(0.0169,0.0179)

4.66

0.0197

(4.64,4.68)

(0.0192,0.0202)

4.63

0.0198

(4.61,4.65)

(0.0193,0.0203)

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Run IV

Shannon index 4.87

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Note: Color should be used for any figures in print

Figure 1. Schematic of the upflow alternating anaerobic/aerobic biofilter system. Influent pump (1), Intermediate pump (2), Biofilter (3), Effluent valve (4), Air compressor (5), P recovery tank

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(6), Intermediate tank (7), Influent tank (8), Programmable controller (9).

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obtained from biofilm sample port S1.

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Figure 2. Florescent and TEM photographs showing the poly-P enriched cells (left column) and PHA enriched cells (right column) in the biomass of AABF obtained at the end of run I (a) - (d), and run IV (e) and (f), respectively.

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Figure 3. PHA percentages in the biomass at each end of P-accumulation cycle or after P recovery (Note that PR in the abscissa stands for P recovery, and A stands for P accumulation).

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Figure 4. The profiles of influent TP and effluent TP and the concentration distribution of various N species in the different operational runs (from run I to run V) by AABF (a)-(e), and the average TP, TN and NH4+-N removal in the different runs (f). Note: AN and AE in the abscissas represent the anaerobic duration and aerobic duration in the alternating anaerobic/ aerobic cycles, respectively.

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Figure 5. The relative abundance of the top 15 genera in the biofilm samples from the different operational runs. The relative abundance for each genus was defined as the sum of OTUs assigned to a genus divided by the total OTUs of a biofilm sample. The phylotypes identified with a relative abundance less than 1% of the total were merged and denoted as “others”.

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Figure 6. The proposed special distribution of PAOs, nitrifiers and denitrifiers (DPAO and DGAO) in the biofilm of AABF and the corresponding biofilm TN and P removal pattern via simultaneous aerobic nitrification and denitrification (SND) and P accumulation.

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P harvesting via carbon source supplement resulted in efficient PHA storage

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Simultaneous PHA-based nitrification/denitrification/P accumulation occurred

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Increased NH4+-N loading did not significantly impact P removal and

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Rapid proliferation of Candidatus Competibacter was observed

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accumulation