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Ultrasonic Treatment Enhanced Ammonia-oxidizing Bacterial (AOB) Activity for Nitritation Process Min Zheng, Yan-chen Liu, Jia Xin, Hao Zuo, Chengwen Wang, and Weimin Wu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04178 • Publication Date (Web): 17 Dec 2015 Downloaded from http://pubs.acs.org on December 17, 2015

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Environmental Science & Technology

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Ultrasonic Treatment Enhanced Ammonia-oxidizing

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Bacterial (AOB) Activity for Nitritation Process

3 4

Min Zheng,† Yan-Chen Liu,*,† Jia Xin‡, Hao Zuo,† Cheng-Wen Wang*,† and Wei-Min Wu§

5

†

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Pollution Control, Tsinghua University, Beijing 100084, China

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‡

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266100, China

9

§

School of Environment, State Key Joint Laboratory of Environment Simulation and

College of Environmental Science and Engineering, Ocean University of China, Qingdao

Department of Civil & Environmental Engineering, the William & Cloy Codiga Resource

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Recovery Research Center, Center for Sustainable Development & Global Competitiveness,

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Stanford University, Stanford, CA94305, USA

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Corresponding Author

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*

Phone: +86 10 6277 1551; Fax: +86 10 6278 8148;

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E-mail addresses: [email protected] (Yan-Chen Liu), [email protected]

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(Cheng-Wen Wang).

17 18

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ACS Paragon Plus Environment


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ABSTRACT

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Oxidation of ammonia to nitrite rather than nitrate is critical for nitritation process for

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wastewater treatment. We proposed a promising approach by using controlled ultrasonic

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treatment to enhance the activity of ammonia-oxidizing bacteria (AOB) and suppress that of

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nitrite-oxidizing bacteria (NOB). Batch activity assays indicated that when ultrasound was

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applied, AOB activity reached a peak level and then declined but NOB activity deteriorated

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continuously as the power intensity of ultrasound increased. Kinetic analysis of relative

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microbial activity versus ultrasonic energy density was performed to investigate the effect of

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operational factors (power, sludge concentration and aeration) on AOB and NOB activities

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and the test parameters were selected for reactor tests. Laboratory sequential batch reactor

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(SBR) was further used to test the ultrasonic stimulus with 8-hour per day operational cycle

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and synthetic waste urine as influent. With specific ultrasonic energy density of 0.09 kJ/mg

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VSS and continuously fed influent containing above 200 mg NH3–N/L, high AOB

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reproductive activity was achieved and nearly complete conversion of ammonia-N to nitrite

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was maintained. Microbial structure analysis confirmed that the treatment changed

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community of AOB, NOB and heterotrophs. Known AOB Nitrosomonas genus remained at

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similar level in the biomass while typical NOB Nitrospira genus disappeared in the SBR

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under ultrasonic treatment and after the treatment was off for 30 days.

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KEYWORDS: Ultrasonic bidirectional stimulation, Kinetics analysis, Nitritation, Rapid

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start-up, Microbial community. 2 / 34


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INTRODUCTION

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Nitrogen removal from wastewater via nitrite i.e. ammonia oxidation to nitrite (or

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nitritation) followed by shortcut denitrification or anaerobic ammonia oxidation

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(ANAMMOX) processes has been attractive for years because of saving energy and carbon

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source required as well as smaller foot print than conventional nitrification followed by

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denitrification process.1-3 This reaction requires conversion of ammonium to nitrite but not

45

further to nitrate by controlling microbial nitrogen metabolic pathway as

NH+4 +1.5O2 → NO-2 +2H+ +H2O

(1)

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Achievement of the nitritation basically depends on selection of microbial population in

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which nitrite-oxidizing bacteria (NOB) is suppressed or eliminated and ammonia-oxidizing

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bacteria (AOB) is predominated.1-3 To date, researchers have proposed several approaches to

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the selection of AOB and suppression of NOB. In 1998, the SHARON process was firstly

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proposed, which employed mesophilic temperature ranging between 30 and 40°C and short

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sludge retention time (SRT) of about 1.5 days to enhance nitritation.4 At present, the common

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methods to achieve nitritation include maintenance of low oxygen, pH control, as well as

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inhibition by free ammonia (FA) and free nitrous acid (FNA).5-7 However, AOB activity may

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also be partially inhibited under the conditions of complete inhibition of NOB activity.8, 9 Full

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scale partial nitritation/anammox experiences indicated that the nitritation process stability is

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critical10. If nitritation were destroyed due to sudden shocks caused by inflow quality/quantity

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variation, operational control failure, or on-site accident etc., the time for regaining efficient 3 / 34



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process performance would be very long. For example, Joss et al. 11 reported that temporarily

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reduced oxygen depletion caused nitritation-anammox process failure as long as 1 month.

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The reason was that after partial loss of AOB biomass and/or activity, the AOB growth in the

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microbial acclimation process was slow. A practicable approach for the restore or start-up of

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nitritation is to use fresh activated sludge (AS) from wastewater treatment plant as inoculum

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or seed sludge. Because AS contains both AOB and NOB, the selection of AOB and

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suppression of NOB from the sludge remains a critical issue.

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Ultrasound is an oscillating sound pressure wave with a frequency greater than the upper

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limit of the human hearing range above 20 kHz. Ultrasonic treatment is mainly applied as

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cleaner and homogenizer in laboratory, to disinfection of water and wastewater, and for the

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enhancement of biodegradability of sludge in anaerobic digesters via destruction of cells.12 In

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addition, research results have reported that ultrasonic treatment could enhance microbial

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productivity13, 14 and stimulate microorganisms for biodegradation, recover active biomass

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from waste activated sludge, enhance phosphorus removal and ANAMMOX activity.15-19

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Previously, researchers found ultrasonic treatment had impact on nitrogen removal in

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wastewater. Zhang et al. reported respective increase in organic, NH4--N, NO2--N and NO3--N

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loads due to the decrease in sludge concentration under ultrasonic operation condition.20

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Research results indicated that nitrifying bacteria (AOB and NOB together) were insensitive

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to ultrasound and denitrification was enhanced quite remarkably by ultrasound.21 Our recent

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studies described that with three different intensities of the ultrasonic treatment impacted the 4 / 34


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overall performance of organic removal, nitrification and denitrification.22 This discovery

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suggested that ultrasonic treatment could provide a promising approach to the selection AOB

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via the enhancement of AOB activity and the suppression of NOB for the start-up of

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nitritation process and maintenance AOB dominance in reactor. We hypothesized the reaction

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as ultrasonic bidirectional effect, which needs more tests.

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The goal of this study was to characterize the ultrasonic bidirectional effect on AOB

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activity and NOB and test the feasibility of the enhancement of AOB and suppression of

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NOB for rapid start-up of nitritation. Our testes included a) the effect of ultrasonic treatment

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factors on major functional microbial activities i.e. AOB, NOB and heterotrophic bacteria; b)

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laboratory-scale sequential batch reactor (SBR) tests for the investigation of the enhancement

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of nitritation by ultrasonic treatment and long-term stability of reactor performance and

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nitritation; and c) microbial community analysis focused on AOB and NOB in the reactor

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biomass. We have performed kinetic analysis of the relative microbial activity as function of

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specific ultrasonic energy density to investigate the effects of operational parameters (energy

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density, sludge concentration and aeration) on the AOB and NOB activities. This analysis

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showed that the data fit well with kinetic equations in the individual ultrasonic batch assays.

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Our results demonstrated that the response of AOB activity to ultrasonic treatment is novel

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and the proper control of the treatment density can enhance AOB activity and suppress NOB

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for rapid start-up of nitritation process.

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MATERIALS AND METHODS 5 / 34



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Batch Assays.

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Batch assays were conducted in volumetric flasks with a volume of 3.0 L in duplicate.

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Conventional nitrification sludge from a full-scale municipal wastewater treatment plant in

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the campus of Tsinghua University (Tsinghua Water Reuse, Beijing) was used as inoculum.

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The sludge was added to the flask, stirred and then continuously treated by an ultrasound

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generator (SCIENTZ–ⅡD, Ningbo Xinzhi Co., Ltd, variable parameters in ranges of 9.5–950

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W, 0–999 min, 20–25 kHz). The mixed liquor samples were taken periodically for microbial

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activity analyses. The activities of AOB, NOB and heterotrophic bacteria of the sludge under

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various ultrasonic intensities, powers, active volumes, and biomass concentrations as well as

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with and without aeration by compressed air were investigated in groups of batch assays, as

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summarized in Table 1.

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Reactor Set-up and Operation.

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Experiments were performed in two sets of laboratory-scale SBRs. One SBR was equipped

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with ultrasound generator (ZJS–1000–500, 100 W, 40 kHz, Hangzhou Success Ultrasonic

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Equipment Co., Ltd) and another was control. The reactors were made from Plexiglas

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cylinder with a height 30 cm and inner diameter 12 cm (effective volume of 2.7 L). The

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reactor was operated with an 8-hour cycle and a hydraulic retention time (HRT) of 1.0 day.

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Each cycle consisted of four periods: water inflow (5 min), aeration (7 h, includes mixing),

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settling (55 min), and decanting (5 min). Airflow rate of 0.6 L/min maintained DO

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concentration over 1.0 mg/L during aeration period. 6 / 34


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The operational conditions of ultrasonic treatment in this study were basically similar to

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that of our previous studies.22 The ultrasound generator started to work after fresh influent

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filled in the reactor during each operation cycle at frequency of 40 kHz and was turned-off

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after 0.5-2.0 hours, depends on tests. The interval time between two ultrasonic treatment was

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set equal to 8-hour operational cycle time. The control reactor was operated without

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ultrasonic treatment. In this study, our tests targeted source-separated human urine as influent,

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which contained average 684.5 ± 54.3 mg/L NH4+-N and 587.5 ± 102.4 mg/L chemical

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oxygen demand (COD) with and pH of 8.8 ± 0.1. The wastewater properties were similar to

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the source-separated urine used in the literature.23 Initially, the reactors were inoculated with

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conventional nitrification sludge from the full-scale municipal wastewater treatment plant.

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The AOB reproductive activities and microbial communities were analyzed during the

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reactors operation.

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Analytical Methods.

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Chemical analysis and calculation. Measurements of COD, NH4+–N, NO2-–N, NO3--N and

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VSS in the reactor liquid phase were performed in the accordance with Standard Methods.24

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DO, pH, and temperature were automatically recorded using a pH/DO meter (WTW,

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pH/Oxi340i). FA and FNA concentrations were calculated as described by Anthonisen et al.8

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The intensity of ultrasonic treatment used was expressed as energy density (ES, kJ/mL) and specific energy density (Espec, kJ/mg VSS) in this study, and calculated using:

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ES =

P⋅t V

ESpec =

(2)

P⋅t V ⋅ VSS

(3)

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where, P is the ultrasonic power (W), t is the irradiation time (s), V is the effective volume of

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the volumetric flask or reactor (L), and VSS is the volatile suspended solid concentration

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which was used express biomass concentration (g/L).

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The nitrite accumulation ratio (NAR) in the effluent of the SBR was calculated as

NAR=

NO−2 − N ×100% NO−2 − N + NO3− − N

(4)

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where NO2--N and NO3--N represent the nitrite-N and nitrate-N concentrations in the effluent,

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respectively.

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Microbial activity assay. The activities of three trophic groups i.e. AOB, NOB and

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heterotrophic bacteria were assayed in batch incubation reactors (active volume 300 mL) and

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expressed in terms of maximum oxygen uptake rate (OURmax, mg O2/(L·h)). The ammonia

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and nitrite oxidation rates were monitored separately by dosing with the selective inhibitors

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allythiourea (ATU) and sodium chlorate (NaClO3) during the respirometer monitoring as

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described by Surmacz-Gorska et al.25 The activities of AOB and NOB were assayed using

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substrates of ammonia (10 mg N/L) and nitrite (10 mg N/L), respectively. The activities of

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heterotrophic bacteria was measured under endogenous respiration conditions.

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AOB reproductive activity assay. The AOB reproductive activity, represented by the 8 / 34


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maximum AOB reproductive rate (µmax - b), was estimated by using aerobic nitrification

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batch experiments mentioned in IAWQ model.26 Sludge sample (5 mL) was withdrawn from

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the reactor and then independently incubated over three days under full aeration (DO ≥ 4.0

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mg/L) in batch reactors, which contained 100 mg/L NH4+-N, 770 mg/L NaHCO3, 155 mg/L

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K2HPO4, 1385 mg/L KH2PO4, and microelement solution described by Zheng et al.27 The

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temperature was controlled at 25 ± 2°C and pH adjusted to 7.25 ± 0.10 with 2.5 N NaOH

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solution. The samples were taken periodically for nitrite and nitrate analysis. The value of the

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rate (µmax - b) was calculated as the slope of curve by plotting natural logarithm of nitrite and

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nitrate nitrogen concentrations versus time.

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DNA extraction. The sludge samples were centrifuged at 5,000 rpm for 4 min before

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extraction of DNA. Fast DNA Spin Kit for Soil (MP Biomedicals, LLC, Solon, OH, USA)

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was used to extract DNA according to manufacturer’s protocol. The extracted DNA was

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purified using ethanol precipitation. The DNA concentration was determined using Nanodrop

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2000 to guarantee values of OD260/OD280 and OD260/OD230 above 1.8 and 2.0,

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respectively. The DNA quality was checked by running a 3-µL DNA solution on a 1% argose

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gel.

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Illumina Miseq sequencing and data analysis. The PCR-amplicons of V4-V5 region of the

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bacteria 16S ribosomal RNA gene with primer 338F and 806R was generated by using the

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extracted DNA as template. The amplicons were sequenced on an Illumina MiSeq platform

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according to the standard protocols. Quatitative Insights Into Microbial Ecology (QIIME) 9 / 34



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pipeline with default settings were used for processing the DNA sequenced data. The

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sequences were clustered into Operational Taxonomic Units (OTUs) using a minimum

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identities of 97% by the UCLUST software. The relative abundance of a genus in community

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of sludge sample was defined as the number of sequences affiliated with that genus divided

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by the total number of sequences in that sludge sample.

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RESULTS AND DISCUSSION

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Effects of Ultrasonic Treatment on Microbial Activities.

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The test conditions of six batch assays are summarized in Table 1. The activities of AOB,

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NOB and heterotrophic bacteria of the test sludge under various intensities, powers and

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biomass concentrations of ultrasonic treatment were normalized with the data of the control

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sludge as shown in Figure 1. No aeration (bubbling compressed air) was applied except for

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the test in Figure 1F. Under all energy levels tested from 100 to 600 W (Figure 1A to 1C),

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AOB activity increased, and reached a peak level as specific energy (Espec) increased. The

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peak was greater than the control by 20-50% at Espec of 0.2-0.3 kJ/mgVSS. Afterwards, the

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activity declined as Espec increased further. The NOB behaved in a similar way when the

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ultrasonic treatment was at low power (100W and 300 W) (Figure 1A and 1B). But it

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declined at the beginning as a high ultrasonic power of 600 W was applied (Figure 1C). The

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results indicated that relatively high power (i.e. 600 W) was needed to suppress NOB activity

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with sludge concentration of about 5000-5500 mg VSS/L.

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Further test indicated that at a decreased sludge concentration (3900 mg VSS/L) more 10 / 34


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favorable results were achieved, and the enhancement of AOB activity and suppression of

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NOB activity was significant at a the same power of 600 W. The peak activity of AOB was

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observed at Espec of 0.2 kJ/mgVSS (Figure 1D).

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As reported previously, aeration in the liquor significantly affected the ultrasonic cavitation

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effect for microbial inactivation.28 The effects of aeration on AOB and NOB activities were

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tested with power of 100 W and sludge concentration of 2270 mg VSS/L (Figure 1E and 1F).

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With aeration, significant enhancement of AOB and depression of NOB activities were

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observed with a low ultrasonic power (i.e., 100 W) and the peak activity was observed at Espec

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of only 0.1 kJ/mgVSS (Figure 1F). As expected, poor results were observed at this low

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energy without aeration (Figure 1E). This confirmed that supply proper aeration is helpful to

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provide selection pressure for AOB and NOB and save energy consumption for ultrasonic

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treatment.

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The impact of ultrasonic treatment on heterotrophic activity showed a slightly similar trend

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to NOB but the effect of the energy density appeared even more significant (Figure 1). For

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instance, as shown in Figure 1D, after the specific energy rose beyond 0.2 kJ/mg VSS, the

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sequence relative activity was AOB > NOB > Heterotrophic bacteria at the same ultrasonic

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energy density, and at a Espec of 0.40 kJ/mg VSS, both AOB and NOB remained only 40% of

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the original activities.

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Kinetic Analysis.

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Based on previous reports12, a kinetic model for the description of the process of ultrasonic 11 / 34



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treatment on microbial activity should include the terms of inactivation and stimulation. The

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intensity of the ultrasound is considered as a viable parameter causing the inactivation of

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microorganisms in homogeneous and heterogeneous mixed cultures in aqueous solution. In

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this study, we modified the process kinetics for aqueous phase ultrasonic disinfection

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developed by Inca and Belen (eq (5)),29 and used specific ultrasonic energy density Espec

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(kJ/mg VSS) to replace the contact time t in the Inca and Belen equation as shown in eq (6)

X = exp( a + b ⋅ t n )

(5)

n

 P ⋅t  n X = exp( a + b ⋅   ) = exp( a + b ⋅ Espec ) V ⋅ VSS inital  

(6)

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where X is the variable in the concentration of microbes with the ultrasonic treatment (mg

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VSS/L), a, b, and n are the model constants (n = 2.5),29 and Espec (kJ/mg VSS) was calculated

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based on the initial concentration of sludge (VSSinital).

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The activation energy is a term to describe the minimum energy which must be available to

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a bio-chemical system with potential reactants to result in a bio-chemical reaction. It was

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reported that ultrasound at certain intensity level enhanced activity of dehydrogenate enzyme

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in bacteria.17, 18, 20 We introduced apparent activation energy Ea as a term for the enhancement

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of enzymatic activity, and used an ultrasonic energy conversion factor f to describe the

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modified apparent activation energy Ea’, as following:

Ea ' = Ea − f ⋅ Espec

(7)

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The maximum oxygen uptake rate OURmax, which is used to describe AOB or NOB activities,

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was calculated based on the IAWQ model in eq (8), and the relationship between maximum

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specific growth rate µmax and apparent activation energy Ea was described by Arrhenius

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equation in eq (9).

OUR max = −

4.57 ⋅ Y ⋅ µmax ⋅ X 1− Y

(8)

 Ea    R ⋅T 

(9)

µmax = A ⋅ exp  −

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The relative activity γ represented by the ratio of OUR’max with ultrasound energy density to

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initial OURmax,initial (without ultrasound) was normalized by the activity in the presence of

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of ultrasonic treatment versus that without ultrasound as:

γ=

OUR max ×100% OUR max,initial

(10)

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where OURmax is the specific activity (mg O2/g VSS·h) of certain trophic bacteria under

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certain ultrasonic energy density, OURmax,initial is the specific activity (mg O2/g VSS·h) of

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certain trophic bacteria without the treatment.

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So, the relative activity γ can be calculated (see supplementary material) as  f  ⋅ E spec + b ⋅ E spec n   R ⋅T 

γ = exp 

(11)

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where, Y is the biomass yield; A is model constant (non-dimensional), T is temperature (T =

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298 K or 25oC in this study), and R is the constant of 8.31 J/(mol·K). 13 / 34



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Using the results from batch assays described latter, the values of OURmax, for AOB and

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NOB were plotted by using nonlinear regression to obtain γ versus Espec to fit the kinetic

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model. The parameter f and b were simultaneously estimated by minimizing the sum of

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squares of deviations between measured γ data and predicted values in the ultrasonic batch

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assays using the software package MATLAB R2014a.

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The tests results of AOB and NOB activities under above six conditions well fit the kinetic

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model by regression plotting (Figure 1). The model constants f and b corresponding to each

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data curve are listed in Table 1. The parameter results showed that the estimated f value for

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AOB was always higher than the f value for NOB, whereas the b value for AOB was

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relatively close or lower than that for NOB. This result indicated that the ultrasonic treatment

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was beneficial to enhancement of AOB activity at low specific energy density and cause both

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AOB and NOB inactivation beyond certain level, which is the function of sludge

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concentration and strongly impacted by aeration. The kinetic modeling can provide a

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powerful tool to screen optimal ranges of operational parameters, which result in relatively

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high f value for AOB and low f and b values for NOB. The data of heterotrophic bacteria did

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not fit the model well (Data not shown). This could be due to complicated population with

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various different responses to ultrasonic selection pressure.

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The results suggested that the model can be used to examine the effect of ultrasonic

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treatment on AOB and NOB activities and help in the selection of the appropriate operational

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parameters to for the enhancement of AOB and depression of NOB at reasonably optimal 14 / 34


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conditions. Based on the results of batch assays, we have selected Espec value below 0.15

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kJ/mg VSS, power value of 100 W, and treatment in aeration for the start-up of nitration

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process in laboratory-scale SBR.

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Start-up of Nitritation Process.

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A SBR was started-up with ultrasonic treatment while another was started as control

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without ultrasound. Initially, both reactors were inoculated with the conventional nitrification

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sludge and continuously fed the synthetic urine influent. The SRT of the SBR was maintained

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at around 100 days. The influent ammonia nitrogen concentration was 669 mg/L and FA

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concentration reached above 200 mg NH3-N/L. Subsequently, the test SBR was treated with

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ultrasound with a set-point ES = 0.09 kJ/mL (or initial Espec = 0.09 kJ/mg VSS). The start-up

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performance of the ultrasonic reactor and control reactor is shown in Figure 2A and 2B,

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respectively. Because FA concentration ranging from 10 to 150 mg NH3-N/L evidently

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inhibited the ammonia oxidization activity,8 the AOB reproductive activities of the sludge in

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the both ultrasonic and control reactors declined initially. The AOB reproductive activity

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started increasing in the ultrasonic reactor after one day, and the NAR in the effluent

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increased and stably maintained above 99% within three days. The effluent nitrite

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concentration was increase sustainably in and stabilized at above 200 mg/L in the ultrasonic

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reactor on day 7. On the other hand, a long lag phase for the recovery of the AOB

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reproductive activity was observed in the control reactor. The AOB activity started increasing

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after about 7 days with appearance of effluent nitrite. The effluent nitrite reached 200 mg/L 15 / 34



280

on day 13. Furthermore, the AOB reproductive activity in the ultrasonic reactor was around

281

twice higher than that in the control reactor. These results indicated that application of the

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ultrasonic treatment did hinder the inhibition of AOB by high FA concentration and rapidly

283

regained high AOB reproductive activity.

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Effect of Ultrasonic Energy Density on Nitritation.

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The ultrasonic reactor was re-inoculated and restarted again to investigate the effect of the

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ultrasonic energy on reactor performance with different set-point specific energy density

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(Figure 3). The influent ammonia-N concentration was 686 ± 32 mg/L. During initial 8 days

288

no ultrasonic treatment was applied. The effluent nitrite-N concentration remained lower than

289

20 mg/L without observed concentration increase. After ultrasonic treatment was applied at

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ES of 0.045 kJ/mL, the effluent nitrite-N increased considerably. The effluent ammonia

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concentration also decreased gradually, indicating that the ultrasonic treatment did stimulate

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AOB activity and their growth as observed in previous test.

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It was reported that ultrasonication in aqueous solution can also oxidize ammonia to nitrite

294

via hydroxyl free radical.30 In order to verify whether the rapid nitrite accumulation was

295

result of biological nitritation instead of chemical oxidation, the ultrasound was applied

296

continuously in the reactor with extremely low oxygen supply (DO < 0.02 mg/L) to inhibit

297

the biological nitritation process from day 11 to day 13 day (see arrow representative in

298

Figure 3). The effluent nitrite concentration decreased swiftly to near zero. On day 13,

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aeration was restored, and the nitrite concentration increased at the same rate as before. This 16 / 34


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result indicated that the nitrite was produced due to stimulated AOB activity but not attributed

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to hydroxyl free radical and confirmed that the nitritation process was accelerated in the

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presence of ultrasound.

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Excessive ultrasonic treatment could cause severe cell damage resulting in sludge floc

304

disintegration, cell lysis and inactivation.31,

32

305

ultrasonic treatment negatively effects on nitritation, a higher level of ES of 0.09 kJ/mL was

306

tested. The results showed that the nitrite concentration remained high in the effluent at this

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energy level. After the ultrasonic treatment was stopped on day 22 to 27, the effluent nitrite

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remained as high as 650 mg/L and ammonia concentration remained near zero. After day 27,

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ES rose to 0.18 kJ/mL and the effluent nitrite dropped gradually with the ammonia

310

accumulation at the same period (Figure 3). On day 35, the effluent nitrite declined to 150

311

mg/L and ammonia rose to 500 mg/L, indicating failure of AOB activity. This result was

312

consistent with the decline of AOB activity under high ultrasonic energy density as observed

313

in batch assay, suggesting that proper ES of 0.09 kJ/mL (around 0.10 kJ/mg VSS) should be

314

employed and a higher level of 0.18 kJ/mL (around 0.20 kJ/mg VSS) should be avoided.

To determine energy limit above which

315

Performance of Stable Operation.

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Another ultrasonic reactor was started with 0.09 kJ/mL and operated continuously for 30

317

days. SRT was controlled at 10 days. Subsequently, the ultrasound was turned-off, the reactor

318

was operated for another 30 days. The operational performance of the reactor before and after

319

turning-off ultrasound for 30 days, including ammonia removal, NAR, specific ammonia 17 / 34



320

removal rate etc., is summarized in Table 2. The nitritation performance was basically

321

unchanged. Both ammonia removal (92.2%) and nitrite in total NOx (99.2%) after 30 days

322

were even slightly higher than those with ultrasonic treatment. COD removal (70-80%) was

323

not relatively very high because the urine wastewater contained small amounts of hardly

324

biodegradable organics, which could not be removed even using long HRT of 2.5 d.24 The

325

major change was the increase in biomass concentration from 1040 ± 30 mg VSS/L at the end

326

of ultrasonic treatment period to 1670 ± 100 mg VSS/L after 30 days without ultrasound. This

327

suggested that observed sludge yield was increased by 37.7% in the absence of ultrasonic

328

treatment. Similar observation of reduction of sludge yield by ultrasound treatment was

329

reported previously by other researchers.33-36 Based on specific activities of AOB and

330

endogenous activity, the biomass was relatively more active in the presence of ultrasonic

331

treatment. The results indicated that the AOB activities remained in the dominance after 30

332

days of turning-off ultrasound, suggesting that ultrasonic treatment is no longer needed as an

333

operational tool after nitritation process is established.

334

Microbial Community Structure.

335

The sludge samples were collected from the SBR reactor of stable operation in the initial

336

fed-batch phase, and during ultrasonic treatment for 30 days and after 30 days of turning-off

337

ultrasonic treatment. The microbial communities were analyzed by using Illumina Miseq

338

sequencing methods. The relative abundances of major bacterial genus on days 1-3, days

339

17-30 and days 49-59 are presented in Figure 4A. The communities generated 162895 DNA 18 / 34


Page 18 of 34

Page 19 of 34


340

gene trimmed sequences, which were separated into 2247 OTUs, from the ten samples. The

341

Good’s Coverage Estimator on the OTUs calculated from each sample showed that this test

342

captured 99% of the species of the samples. The sequencing analysis showed that

343

Nitrosomonas genus, which is known AOB, was found in the inoculated sludge with relative

344

abundance of 5.82% ± 3.01% and still high level with relative abundance of 4.98% ± 1.55%

345

in the biomass receiving 30 days of ultrasonic treatment. This organism remained

346

predominant as 5.01% ± 1.61% in the biomass after ultrasonic treatment was turned off for 30

347

days. The genus of Nitrospira, a typical NOB, was detected only in the inoculated sludge but

348

was not detected in the reactor during ultrasonic treatment and afterwards, indicating that it

349

was completely eliminated after the nitritation process was established and could not remerge

350

after the ultrasonic treatment was no longer applied.

351

Other than AOB and NOB, the ultrasonic treatment changed microbial diversity, i.e. the

352

relative abundances of most heterotrophic bacteria significantly declined in the ultrasonic

353

period. However, the application of ultrasound positively affected the growth of some

354

heterotrophic genera such as Arenimonas, Thauera and Weeksella. Their relative abundances

355

reached as high as 24.58% ± 9.34%, 11.29% ± 10.74% and 8.97% ± 5.34%, respectively.

356

However, the abundances of three genera deceased after the ultrasonic treatment stopped.

357

Thauera spp. was considered as a critical population for organic load degradation in many

358

wastewater treatment plants.37 The ultrasonic treatment induced the death of a large number

359

of heterotrophic bacteria and the releasing of intracellular organic matters into the mixed 19 / 34



360

liquor.31 This might be very conducive to the rapid growth of Thauera spp. under the

361

conditions of ultrasonic treatment. Arenimonas, Thauera and Weeksella belong to organic

362

carbon-degrading bacteria but their roles are not clear in detail.

363

The ultrasonic treatment had significant impact on microbial community structure. The

364

most promising observation was the high level of AOB abundance and elimination of NOB.

365

However, the diversity of heterotrophic bacteria appeared increasing after ultrasonic

366

treatment was terminated. Consistently, PCA analysis showed that microbial community

367

structures were clustered by ultrasonic treatment (Figure 4B). The communities at the

368

beginning (day 1, 3 and 6) can be clustered as a group with those after ultrasonic treatment

369

was terminated (day 49 and day 59). The PCA analysis suggested that the communities in all

370

samples during ultrasonic treatment can be grouped together.

371

Implementation.

372

The response of AOB and NOB to ultrasonic treatment showed bidirectional effect i.e. the

373

AOB activity was enhanced, reached a peak level, and then declined while the NOB activity

374

declined as ultrasonic intensity increased at low frequency of 20-40 kHz. In this study, we

375

identified that this effect was influenced by ultrasonic power applied, sludge concentration,

376

and supply of aeration. A kinetic model was developed and fit the batch assay data well.

377

Based on the results of batch assays, the nitritation process was successfully started-up with

378

inoculation of the nitrification sludge within three days even with treatment of high-strength

379

ammonium wastewater which could cause strong FA inhibition on AOB activity at high 20 / 34


Page 20 of 34

Page 21 of 34


380

ammonia sludge loading. The AOB predominance in reactor biomass could last more than 30

381

days without NOB activity in the laboratory reactor test. Microbial community analysis of the

382

reactor biomass confirmed the AOB predominance. The results revealed that ultrasonic

383

treatment can serve as a promising approach to the start-up of nitritation process by

384

enhancing AOB activity and suppressing NOB in inoculated sludge and reactor biomass. This

385

approach is easier to be operated and controlled in comparison with previously proposed

386

control methods for achieving nitrite accumulation by limiting oxygen level, high FA and

387

FNA inhibition, short SRT as well as high temperature (up to 35 oC) to suppress NOB activity

388

in biomass.5-7 The finding of this study is a breakthrough of selection of AOB population with

389

depression of NOB.

390

The results of this study indicated that a properly controlled ultrasonic intensity is essential

391

to enhance the AOB and depress NOB activities. In addition, aeration and sludge

392

concentration had strong impact on the bidirectional effect. Although we identified a proper

393

ultrasonic density level in this study, due to scale effect, further research will be needed to

394

test the various ultrasonic power density/intensity with at least pilot scale reactor to obtain

395

sound operational parameters for full scale application. The air loading and bubble size could

396

also influence the performance. The economical assessment of ultrasonic technique for

397

full-scale implementation should be evaluated. The full application of the ultrasonic treatment

398

for nitritation process will address on all operational factors, including density/intensity,

399

specific energy input, and ultrasonic frequency, duration of ultrasonic treatment (i.e. partial 21 / 34



Page 22 of 34

400

and complete ultrasonic treatment), aeration method and strength, biomass concentration, and

401

different sludge source on the ultrasonic processing of sludge. Currently, ultrasonic

402

equipments for sludge treatment are commercially available28 with flow rates from 0.02 to

403

200 m3/h, which can be selected or modified for the nitritation process. On the other hand,

404

ultrasonic cavitation plays a main role in microbial inactivation, and influenced by many

405

environmental factors, such as increasing ultrasonic density, intensity, and decreasing

406

particulate

407

gram-negative (P. fluorescens), rod-shape bacteria were more susceptible to the ultrasonic

408

treatment than gram-positive (S. thermophiles), coccus-shaped bacteria,38 whereas some

409

reported that there was no differences in resistance to ultrasound between gram-negative (P.

410

aeruginosa and E. coli) and gram-positive (S. aureus and B. subtilis) bacteria.39 In this study,

411

bacterial genes belong to Nitrosomonas (AOB) and Nitrospira (NOB) were found in the

412

inoculated sludge. Both are gram-negative bacteria, and no significant physiological traits

413

differences have been reported between them.1 The morphological feature did not seem to be

414

a differentiating factor in ranking the organisms by percent killed by ultrasonic treatment. The

415

target of ultrasonic damage might be the inner (cytoplasmic) membrane, which consists of a

416

lipoprotein bilayer.39 More research is needed to understand the difference in the ultrasonic

417

tolerance between AOB and NOB and cost effectiveness of ultrasonic treatment by using

418

pilot-scale test.

419

AUTHOR INFORMATION

matter concentration etc.28

Previously,

some

22 / 34


publications found

that

Page 23 of 34


420

Corresponding Author

421

*

422

E-mail addresses: [email protected] (Yan-Chen Liu), [email protected]

423

Phone: +86 10 6277 1551; Fax: +86 10 6278 8148;

(Cheng-Wen Wang).

424

Notes

425

The authors declare no competing financial interest.

426

ACKNOWLEDGEMENTS

427

The research by Dr. Min Zheng was supported by China Postdoctoral Science Foundation

428

funded project 2015T80098. Dr. Wei-Min Wu at Stanford University was a non-funded

429

collaborator in this study.

430

Available Supporting Information

431

Additional information as noted in the text. This material is available free of charge via the

432

Internet at http://pubs.acs.org.

433

REFERENCES

434 435 436 437 438 439 440 441

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The SHARON process: An innovative method for nitrogen removal from ammonium-rich waste water. Water Sci. Technol. 1998, 37 (9), 135-142. (5) Park, S.; Bae, W.; Rittmann, B. E. Operational boundaries for nitrite accumulation in nitrification based on minimum/maximum substrate concentrations that include effects of oxygen limitation, pH, and free ammonia and free nitrous acid inhibition. Environ. Sci. Technol. 2010, 44 (1), 335-342. (6) Ma, Y.; Peng, Y.; Wang, S.; Yuan, Z.; Wang, X. Achieving nitrogen removal via nitrite in a pilot-scale continuous pre-denitrification plant. Water Res. 2009, 43 (3), 563-72. (7) Wang, Q.; Ye, L.; Jiang, G.; Hu, S.; Yuan, Z. Side-stream sludge treatment using free nitrous acid selectively eliminates nitrite oxidizing bacteria and achieves the nitrite pathway. Water Res. 2014, 55, 245-55. (8) Anthonisen, A. C.; Loehr, R. C.; Prakasam, T. B. S.; Srinath, E. G. Inhibition of nitrification by ammonia and nitrous-acid. Journal Water Pollution Control Federation. 1976, 48 (5), 835-852. (9) Vadivelu, V. M.; Keller, J.; Yuan, Z. Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture. Biotechnol. Bioeng. 2006, 95 (5), 830-9. (10) Lackner, S.; Gilbert, E. M.; Vlaeminck, S. E.; Joss, A.; Horn, H.; van Loosdrecht, M. C. M. Full-scale partial nitritation/anammox experiences - An application survey. Water Res. 2014, 55, 292-303. (11) Joss, A.; Derlon, N.; Cyprien, C.; Burger, S.; Szivak, I.; Traber, J.; Siegrist, H.; Morgenroth, E. Combined nitritation-anammox: advances in understanding process stability. Environ. Sci. Technol. 2011, 45 (22), 9735-9742. (12) Tyagi, V. K.; Lo, S. L.; Appels, L.; Dewil, R. Ultrasonic Treatment of Waste Sludge: A Review on Mechanisms and Applications. Crit. Rev. Env. Sci. Technol. 2014, 44 (11), 1220-1288. (13) Rokhina, E. V.; Lens, P.; Virkutyte, J. Low-frequency ultrasound in biotechnology: state of the art. Trends Biotechnol. 2009, 27 (5), 298-306. (14) Chisti, Y. Sonobioreactors: using ultrasound for enhanced microbial productivity. Trends Biotechnol. 2003, 21 (2), 89-93. (15) Schlafer, O.; Onyeche, T.; Bormann, H.; Schroder, C.; Sievers, M. Ultrasound stimulation of micro-organisms for enhanced biodegradation. Ultrasonics. 2002, 40 (1-8), 25-29. (16) Sears, K. J.; Alleman, J. E.; Gong, W. L. Feasibility of using ultrasonic irradiation to recover active biomass from waste activated sludge. J. Biotechnol. 2005, 119 (4), 389-399. (17) Xie, B. Z.; Wang, L.; Liu, H. Using low intensity ultrasound to improve the efficiency of biological phosphorus removal. Ultrason. Sonochem. 2008, 15 (5), 775-781. (18) Yu, J. J.; Chen, H.; Zhang, J.; Ji, Y. X.; Liu, Q. Z.; Jin, R. C. Enhancement of ANAMMOX activity by low-intensity ultrasound irradiation at ambient temperature. Bioresour. Technol. 2013, 142, 693-696. (19) Duan, X. M.; Zhou, J. T.; Qiao, S.; Wei, H. F. Application of low intensity ultrasound to 24 / 34


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enhance the activity of anammox microbial consortium for nitrogen removal. Bioresour. Technol. 2011, 102 (5), 4290-4293. (20) Zhang, R. N.; Jin, R. F.; Liu, G. F.; Zhou, J. T.; Li, C. L. Study on nitrogen removal performance of sequencing batch reactor enhanced by low intensity ultrasound. Bioresour. Technol. 2011, 102 (10), 5717-5721. (21) Xie, B. Z.; Liu, H. Enhancement of Biological Nitrogen Removal from Wastewater by Low-Intensity Ultrasound. Water. Air. Soil Pollut. 2010, 211 (1-4), 157-163. (22) Zheng, M.; Liu, Y. C.; Xu, K. N.; Wang, C. W.; He, H.; Zhu, W.; Dong, Q. Use of low frequency and density ultrasound to stimulate partial nitrification and simultaneous nitrification and denitrification. Bioresour. Technol. 2013, 146, 537-542. (23) Udert, K. M.; Kind, E.; Teunissen, M.; Jenni, S.; Larsen, T. A. Effect of heterotrophic growth on nitritation/anammox in a single sequencing batch reactor. Water Sci. Technol. 2008, 58 (2), 277-284. (24) Ministry Of Environmental Protection, P. R. C. Monitoring and Analytical Methods of Water and Wastewater. 4th Edition ed.; China Environmental Science Press: Beijing, 2006. (25) SurmaczGorska, J.; Gernaey, K.; Demuynck, C.; Vanrolleghem, P.; Verstraete, W. Nitrification monitoring in activated sludge by oxygen uptake rate (OUR) measurements. Water Res. 1996, 30 (5), 1228-1236. (26) Henze, M.; Grady, C. P. L. J.; Gujer, W.; Marais, G. v. R.; Matsuo, T. Activated sludge model No. 1. IAWPRC Scientific and Technical Report No. 1, IAWPRC, London. 1987. (27) Zheng, M.; Liu, Y. C.; Wang, C. W.; Xu, K. N. Study on enhanced denitrification using particulate organic matter in membrane bioreactor by mechanism modeling. Chemosphere. 2013, 93 (11), 2669-2674. (28) Pilli, S.; Bhunia, P.; Yan, S.; LeBlanc, R. J.; Tyagi, R. D.; Surampalli, R. Y. Ultrasonic pretreatment of sludge: a review. Ultrason. Sonochem. 2011, 18 (1), 1-18. (29) Ince, N. H.; Belen, R. Aqueous phase disinfection with power ultrasound: Process kinetics and effect of solid catalysts. Environ. Sci. Technol. 2001, 35 (9), 1885-1888. (30) Fischer, C. H.; Hart, E. J.; Henglein, A. Ultrasonic irradiation of water in the presence of 18,18 O2 - Isotope exchange and isotopic distribution of H2O2. J. Phys. Chem. 1986, 90 (9), 1954-1956. (31) Zhang, P. Y.; Zhang, G. M.; Wang, W. Ultrasonic treatment of biological sludge: Floc disintegration, cell lysis and inactivation. Bioresour. Technol. 2007, 98 (1), 207-210. (32) Rai, C. L.; Struenkmann, G.; Mueller, J.; Rao, P. G. Influence of ultrasonic disintegration on sludge growth reduction and its estimation by respirometry. Environ. Sci. Technol. 2004, 38 (21), 5779-5785. (33) Mohammadi, A. R.; Mehrdadi, N.; Bidhendi, G. N.; Torabian, A. Excess sludge reduction using ultrasonic waves in biological wastewater treatment. Desalination. 2011, 275 (1-3), 67-73. (34) Zhang, G. M.; Zhang, P. Y.; Yang, J. M.; Chen, Y. M. Ultrasonic reduction of excess sludge from the activated sludge system. J. Hazard. Mater. 2007, 145 (3), 515-519. (35) Zhang, G. M.; He, J. G.; Zhang, P. Y.; Zhang, J. Ultrasonic reduction of excess sludge 25 / 34



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from activated sludge system II: Urban sewage treatment. J. Hazard. Mater. 2009, 164 (2-3), 1105-1109. (36) He, J. G.; Wan, T. A.; Zhang, G. M.; Yang, J. Ultrasonic reduction of excess sludge from activated sludge system: Energy efficiency improvement via operation optimization. Ultrason. Sonochem. 2011, 18 (1), 99-103. (37) Mao, Y.; Zhang, X.; Yan, X.; Liu, B.; Zhao. L. Development of group-specific PCR-DGGE fingerprinting for monitoring structural changes of Thauera spp. in an industrial wastewater treatment plant responding to operational perturbations. J. Microbiol. Meth. 2008, 75, 231-236. (38)Villamiel, M.; Jong, P. de. Inactivation of Pseudomonas fluorescens and Streptococcus thermophilus in trypticase soy broth and total bacteria in milk by continuous-flow ultrasonic treatment and conventional heating. J. Food. Eng. 2000, 45, 171-179. (39)Scherba, G.; Weigel, R.M.; O'Brien W.D. Quantitative assessment of the germicidal efficacy of ultrasonic energy. Appl. Environ. Microb. 1991, 57 (7), 2079-2084.

26 / 34


Page 26 of 34

Page 27 of 34


539

Table 1. Batch assays performed under six ultrasonic treatment conditions and kinetic

540

constants for AOB and NOB obtained.

Tests conditions

AOB

Power (W)

Volume (L) VSS (mg/L)

f

1

100

1.5

4890

2

300

1.5

3

600

4

NOB

No. R2

f

b

R2

5314 -4.7

0.969

4212

-4.6

0.954

5370

3180 -2.9

0.992

2432

-2.5

0.985

1.5

5470

2073 -2.3

0.865

0

-5.7

0.972

600

1.5

3900

8823 -19.8

0.975

0

-7.0

0.945

5

100

2.0

2270

2682 -2.3

0.993

468

-1.9

0.995

6*

100

2.0

2270

6087 -30.7

0.991

518

-21.1

0.957

b

541

Note: The constants f and b are obtained by plotting data using equation (9). *Test No. 6 was

542

conducted with aeration. Test sludge was obtained from a full-scale municipal wastewater

543

treatment plant.

544

27 / 34



545

Page 28 of 34

Table 2. Reactor performance during and after ultrasonic treatment.

Parameters

Period I

Period II

Energy density ES (kJ/mL)

0.09

No

Temperature (°C)

26.1 ± 1.6

23.3 ± 0.4

Dissolved oxygen (mg O2/L)

2.38 ± 1.91

3.48 ± 3.03

pH

7.71 ± 0.58

8.04 ± 0.40

Biomass concentration (mg VSS/L)

1040 ± 30

1670 ± 100

Ammonia removal (%)

84.8 ± 9.1

92.2 ± 8.3

Nitrite in total NOx (%)

98.5 ± 0.5

99.2 ± 0.3

Specific maximum ammonia oxidation activity (mg O2/(g VSS·h))

30.4 ± 4.7

27.1 ± 3.2

Specific ammonia removal rate (mg N/(g VSS·h))

24.5 ± 1.0

23.8 ± 0.8

Organics removal (%)

69.3 ± 11.4

79.7 ± 5.0

Specific endogenous respiration activity (mg O2/(g VSS·h))

4.4 ± 2.3

3.1 ± 1.6

Nitritation process

Heterotrophic process

546

Note: Period (I): the average data of 30 days during ultrasonic treatment. Period (II): average

547

data after the treatment stopped for 30 days.

548 549

28 / 34


Page 29 of 34


550

Figure Captions

551

Figure 1. Effect of specific energy density of ultrasonic treatment on the relative activities of

552

AOB, NOB, and heterotrophic bacteria, which were measured by batch assays. The curves

553

are data fitting to model equation (9). The test conditions of A,B.C.D.E and F are listed in

554

Table 1 as Test 1,2,3,4,5 and 6. In 1F, aeration was performed with air flow of 0.6 L/min.

555 556

Figure 2. Comparisons of nitrite accumulation ratio (NAR) and NOx--N (NO2--N + NO3--N)

557

concentrations in the effluent, and reproductive activity of AOB during the start-up period of

558

an ultrasonic (A) and control (B) reactors.

559 560

Figure 3. Performances of nitritation process with ultrasonic treatment at different energy

561

densities (ES). Influent ammonia-N concentration was 686 ± 32 mg/L. The arrow represents

562

the application extremely low oxygen supply (DO < 0.02 mg/L) for three days.

563 564

Figure 4. A: Comparison of relative abundances of major bacterial genera in the sludge

565

collected from the reactor of the initial fed-batch phase for 8 days, the sludge operated under

566

ultrasonic treatment for 30 days and the sludge in the same reactor after ultrasonic treatment

567

stopped for 30 days. The reactor was operated at energy density ES of 0.09 kJ/mL. B:

568

Ordination plot produced from principal component analysis (PCA) of all of detected genus.

569

Circles represent samples collected from the reactor of initial fed-batch phase (d1, d3 and d6),

570

during ultrasonic treatment (d17, d24, d30 and d37) and after the treatment stopped (d49 and

571

d59), respectively.

572

29 / 34



A

140

Page 30 of 34

B

120

γ (%)

100 80 60 40 20 0

AOB NOB Heterotroph

P = 100W VSS = 4890 mg/L

P = 300W VSS = 5370 mg/L

C

140

D

120

γ (%)

100 80 60 40 20 0

P = 600W VSS = 5470 mg/L

P = 600W VSS = 3900 mg/L

F

E

140 120

Aeration P = 100 W VSS = 2270 mg/L

γ (%)

100 80 60 40 20

573

P = 100 W VSS = 2270 mg/L

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.70.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Espec (kJ/mg VSS) Espec (kJ/mg VSS)

574

Figure 1. Effect of specific energy density of ultrasonic treatment on the relative activities of

575

AOB, NOB, and heterotrophic bacteria, which were measured by batch assays. The curves

576

are data fitting to model equation (9). The test conditions of A,B.C.D.E and F are listed in

577

Table 1 as Test 1,2,3,4,5 and 6. In 1F, aeration was performed with air flow of 0.6 L/min.

578

30 / 34


A 0.8

150

0.6

100

0.4 NAR Eff NOx -N (µmax- b)AOB

50

0.2

0

0.0

200

B 0.8

150

0.6

100

0.4

50

0.2

0

0.0 0

579

(µmax- b)AOB (1/d)

-

NOx -N (mg/L), NAR (%)

200

(µmax- b)AOB (1/d)


NOx -N (mg/L), NAR (%)

Page 31 of 34

3

6 9 Time (d)

12

15

580

Figure 2. Comparisons of nitrite accumulation ratio (NAR) and NOx--N (NO2--N + NO3--N)

581

concentrations in the effluent, and reproductive activity of AOB during the start-up period of

582

an ultrasonic (A) and control (B) reactors.

583

31 / 34



Effluent N-concentrations (mg/L)

800

ES = 0

0.045

0.09

0

Page 32 of 34

0.18 kJ/mL

600

400 +

Eff NH4 -N

200

-

Eff NO2 -N -

Eff NO3 -N

0 0

5

10

15 20 Time (d)

25

30

35

584 585

Figure 3. Performances of nitritation process with ultrasonic treatment at different energy

586

densities (ES). Influent ammonia-N concentration was 686 ± 32 mg/L. The arrow represents

587

the application extremely low oxygen supply (DO < 0.02 mg/L) for three days.

588

32 / 34


Page 33 of 34


Relative abundance (%)

70 60 50

Initial fed-batch phase During ultrasonic treatment After the treatment stopped

A

40 30 20 10 0 as ra as ra la m k us d as ia ra as er k as rs on ospi mon haue ksel teriu oran cocc lture mon ttow uepe mon bact oran mon Othe m tr ni T ee ac 9_n ara ncu mo O Tr do oro 5_n ili o i s F W eob 10 P _u er eu h 4 tro N Are e Th s APs Diap -CM Ni a y e r F Ch SH rac -K spi 30 o r G p J Sa

B

589 590

Figure 4. A: Comparison of relative abundances of major bacterial genera in the sludge

591

collected from the reactor of the initial fed-batch phase for 8 days, the sludge operated under

592

ultrasonic treatment for 30 days and the sludge in the same reactor after ultrasonic treatment

593

stopped for 30 days. The reactor was operated at energy density ES of 0.09 kJ/mL. B:

594

Ordination plot produced from principal component analysis (PCA) of all of detected genus.

595

Circles represent samples collected from the reactor of initial fed-batch phase (d1, d3 and d6),

596

during ultrasonic treatment (d17, d24, d30 and d37) and after the treatment stopped (d49 and

597

d59), respectively. 33 / 34



598

Graphic for manuscript

599 600

Abstract graphic

601

34 / 34


Page 34 of 34

Ultrasonic Treatment Enhanced Ammonia ... - ACS Publications

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