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Molecular and kinetic characterization of planktonic Nitrospira spp. selectively enriched from activated sludge Mee-Rye Park, Hongkeun Park, and Kartik Chandran Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05184 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 30, 2017

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Environmental Science & Technology

Title :

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Molecular and kinetic characterization of planktonic Nitrospira spp. selectively enriched

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from activated sludge

4 5 6

Authors : Mee-Rye Park1, Hongkeun Park1 and Kartik Chandran1,*

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Affilation : 1

Department of Earth and Environmental Engineering, Columbia University, 500 West

120th Street, New York, New York 10027, USA.

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Correspondent footnote :

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Kartik Chandran, Department of Earth and Environmental Engineering, Columbia

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University, 500 West 120th Street, New York, New York 10027. Phone : (212) 854 9027,

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email : [email protected]

16 17 18

* To whom all correspondence should be addressed.

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ACS Paragon Plus Environment


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Abstract

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Nitrospira spp. are chemolithoautotrophic nitrite oxidizing bacteria (NOB), which are ubiquitous

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in natural and engineered environments. However, there exist few independent biokinetic studies

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on Nitrospira spp., likely because their isolation and selective enrichment from environmental

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consortia such as activated sludge can be challenging. Herein, planktonic Nitrospira spp. cultures

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closely related to Candidatus Nitrospira defluvii (Nitrospira lineage I) were successfully

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enriched from activated sludge in a sequencing batch reactor by maintaining sustained limiting

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extant nitrite and dissolved oxygen concentrations. Morphologically, the enrichment consisted

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largely of planktonic cells with an average characteristic diameter of 1.3 ± 0.6 µm. Based on

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respirometric assays, estimated maximum specific growth rate (µmax), nitrite half saturation

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coefficient (KS), oxygen half saturation coefficient (KO) and biomass yield coefficient (Y) of the

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enriched cultures were 0.69 ± 0.10 d-1, 0.52 ± 0.14 mg-N/L, 0.33 ± 0.14 mg-O2/L and 0.14 ±

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0.02 mg-COD/mg-N, respectively. These parameters collectively reflect not just higher affinities

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of this enrichment for nitrite and oxygen, respectively, but also a higher biomass yield and

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energy transfer efficiency relative to Nitrobacter spp. Used in combination, these kinetic and

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thermodynamic parameters can help towards the development and application of energy efficient

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biological nutrient removal processes through effective Nitrospira out-selection.

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Keywords: nitrite oxidizing bacteria; Nitrospira spp.; planktonic; kinetics; half saturation

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coefficient; maximum specific growth rate; out-selection; anammox

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1. Introduction

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Nitrite-oxidizing bacteria (NOB) play an important role in natural and engineered nitrogen

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cycling systems including conventional biological nutrient removal (BNR) processes. In recent

43

years, alternatives to conventional BNR processes such as anaerobic ammonium oxidation

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(anammox) and nitritation-denitritation have been more widely applied owing to the energy and

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chemical savings that they offer

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oxidation of ammonium partially to nitrite by ammonia-oxidizing bacteria (AOB), in parallel

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with

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chemoorganoheterotrophic nitrogen removal.

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Among the different NOB found in wastewater treatment systems, Nitrospira spp. and

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Nitrobacter spp. represent the most dominant NOB in terms of diversity and populations

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Recently discovered Nitrotoga have been also regarded as important NOB in full-scale

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wastewater treatment systems

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detect only NOB. However, Nitrotoga were competitive with Nitrospira and Nitrobacter during

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long-term cultivation at 5 and 10 °C, respectively 6, 9.

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Specifically, those related to Nitrospira spp. are relatively more abundant in mainstream

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processes (treating sewage streams) than the more conventionally studied NOB related to

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Nitrobacter spp. and Nitrotoga proliferating at low temperature. Therefore, energy efficient

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process alternatives to conventional mainstream BNR need to accomplish out-selection of

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Nitrospira spp. rather than other NOB. Commonly used limiting factors (e.g. free ammonia and

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free nitrous acid) applied for NOB out-selection10, 11 in sidestream reactors may not be relevant

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to selectively suppress Nitrospira spp. in mainstream reactors because of the low extant free

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ammonia concentrations therein as well as possibly distinct biokinetic characteristics and

NOB

out-selection,

1, 2

. Energy efficiency of these processes is engendered by the

followed

5-8

directly

by

either

chemolithoautotrophic

or

3, 4

.

, where they often coexist with Nitrospira but occasionally



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sensitivities of Nitrospira spp. compared to Nitrobacter spp. Given such challenges, in

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mainstream systems, the strategies for NOB out-selection and stable nitritation have necessitated

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a combination of several factors such as intermittent aeration, and controlled COD input, coupled

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with solids retention time (SRT) control

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Accordingly, fundamental studies of Nitrospira spp. physiology and kinetics are essential to

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inform NOB out-selection under mainstream wastewater treatment conditions. However, such

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studies are exceedingly rare owing to the frequent proliferation of Nitrobacter spp. in engineered

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NOB reactors or enrichments. Some previous laboratory studies have attempted to overcome

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such challenges by enriching Nitrospira spp. through serial dilution with additional selective

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pressure of amendment with antibiotics 14. However, such approaches are not entirely applicable

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to engineered wastewater treatment processes either from a kinetic or physiological perspective.

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Nitrospira spp. have been reported to be sensitive to the concentrations of their substrates, nitrite

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and oxygen. The growth of Nitrospira spp. was inhibited at high nitrite concentrations (above 80

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mg-N/L in bulk liquid), where Nitrobacter spp. were selected

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and low dissolved oxygen (DO) below 0.5 mg-O2/L favored the competition of Nitrospira spp.

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over Nitrobacter spp. 16

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Here, we hypothesized that Nitrospira spp. can be enriched from a mixed environmental

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microbial consortium such as activated sludge through long-term continuous reactor operation

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under limiting extant nitrite and DO concentrations. If successful, such an enrichment would

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provide more representative knowledge on the eco-physiology, metabolism and kinetics under

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conditions similar to that in activated sludge processes. Accordingly, the objectives of this study

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were to (1) selectively enrich Nitrospira spp. from activated sludge in a continuously operated

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sequencing batch reactor (SBR) (2) characterize the microbial ecology of the SBR during the

2, 12, 13

.

1, 4, 15


. Both long SRT (40 days)

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course of enrichment and (3) determine key kinetic parameters including the maximum specific

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growth rate (µmax), oxygen half saturation coefficient (KO), nitrite half saturation coefficient (KS)

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and thermodynamic parameters including the biomass yield coefficient (Y) and energy transfer

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efficiency (ε), describing the Nitrospira spp. enrichment.

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2. Materials and Methods

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2.1. Reactor setup and operation

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Planktonic Nitrospira spp. cultures were cultivated in a lab-scale SBR with a working volume of

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6 L, hydraulic retention time (HRT) of 0.5 d and a target SRT of 15 d. The SBR was seeded with

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activated sludge from the Blue Plains advanced wastewater treatment plant (Washington, D.C.)

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and was operated at four 6 h cycles per day, each comprising 5 h continuous feed and react, 0.75

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h settle, and 0.25 h decant phases. The SBR phases were automatically controlled via a digital

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controller (Chrontrol Corp., San Diego, CA). SRT was controlled by manually withdrawing

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biomass from the SBR at the end of the feed and react phase. The influent concentration of nitrite,

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which served as the sole energy source during the period of operation, was increased

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successively through four phases in the following sequence: 40 ± 1.20 mg-N/L (phase 1, days 0

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to 23), 100 ± 1.73 mg-N/L (phase 2, days 24 to 57), 160 ± 2.38 mg-N/L (phase 3, days 58 to 99),

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and finally 200 ± 1.81 mg-N/L (phase 4, days 100 to 222). The SBR was operated with cyclic

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aeration (2 min air-on and 3 min off) to maintain extant reactor DO concentrations between 0.5 -

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1 mg-O2/L during the feed and react phases by facilitating oxygen diffusion into the liquid by

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gentle mixing with a magnetic stirrer. DO was monitored in real-time using online respirometry

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using Clark-type electrodes (YSI 5331, Yellow Springs, OH), interfaced to an online DO meter

108

(YSI 5300A, Yellow Springs, OH) and to a personal computer. The SBR was operated at room



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temperature (22 ± 1 °C). The feed medium contained the following constituents per liter of tap

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water: 0.005 g (NH4)2SO4; 0.2 - 1 g NaNO2; 0.007 g CaCO3; 0.5 g NaCl; 0.05 g MgCl2 · 6H2O;

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0.15 g KH2PO4; 33.8 µg MnSO4 · H2O; 49.4 µg H3BO3; 43.1 µg ZnSO4 · 7H2O; 37.1 µg (NH4)6

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Mo7O24·4H2O; 25.0 µg CuSO4·5H2O; 973.0 µg FeSO4· 7H2O

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controlled at 7.5 ± 0.1 with 1M NaHCO3. Reactor samples were collected right before the settle

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phase of the SBR. 15 ml samples from the reactor and influent medium tank were collected in

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duplicates and centrifuged at 8,000 x g for 10 min. The concentrations of nitrite (diazotization

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colorimetry) and nitrate (ion selective electrode, Fisher, Waltham, MA) in the supernatant were

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used to monitor the SBR performance. Ion Chromatography (Thermo Scientific Dionex ICS-

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2100., Sunnyvale, CA) was also used to measure nitrite and nitrate concentrations during

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intensive 6 h profiles obtained during individual SBR cycles. Total reactor biomass and effluent

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biomass concentrations were approximated using total chemical oxygen demand (tCOD)

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measurements (Hach Chemical Co., Loveland, CO) owing to the absence of organic carbon in

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the SBR feed.

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2.2. DNA extraction and quantification

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DNA was extracted from biomass samples using a DNeasy Blood & Tissue kit (Qiagen, Inc.,

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Germantown, MD). The concentrations of Nitrospira spp., Nitrobacter spp., and AOB were

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quantified in triplicate via SYBR green chemistry quantitative polymerase chain reaction (qPCR),

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specifically targeting Nitrospira and Nitrobacter 16S rRNA genes, and ammonia

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monooxygenase subunit A (amoA) gene, respectively. Total bacterial concentrations were also

129

quantified using eubacterial 16S rRNA gene targeted primers (Table 1). Standard curves for

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qPCR were generated via serial decimal dilutions of plasmid DNA containing specific target


17

. The pH was automatically

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gene inserts. The absence of primer-dimer was confirmed via melt curve analysis on every qPCR

132

assay conducted (data not shown).

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2.3. Next generation sequencing of amplicon library and sequence analysis

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DNA extracts were purified using QIAquick DNA Cleanup kit with QIAcube (Qiagen, CA). The

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quality and quantity of DNA were checked using NanoDrop Lite Spectrophotometer

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(Thermofisher, MA). Barcoded fusion primers with sequencing adaptors and 1055F/1392R

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universal primer set were applied in each sample for multiplex sequencing. Prior to template

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preparation, library quantification was performed with KAPA Library Quantification kit for Ion

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Torrent (KAPA biosystems, MA) to avoid polyclonality of Ion Sphere Particles (ISP). Template

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preparation with DNA library followed by ISP enrichment was performed using the Ion

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OneTouch2 system following the manufacturer’s instruction (Ion OT2 400 kit, Product No.

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4479878). The enriched ISP was loaded onto an Ion Torrent 316 chip and run on an Ion Torrent

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PGM™ according to manufacturer’s instructions (Ion PGM™ Sequencing 400 Kit, Product No.

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4482002). Ion Torrent Suite software ver. 4.0.2 was used for base calling, signal processing and

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quality filtering (> Phred score of 15) of the raw sequences. Mothur software was applied for

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post-run bioinformatic analysis of the amplicon sequences 18. Relative microbial abundance was

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mapped using heatmap modules in “R” software and phylogenetic affiliation among community

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members was inferred using the neighbor-joining method on Molecular Evolutionary Genetics

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Analysis 6.0 (MEGA 6.0) software

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computational model. Operational taxonomy units (OTUs) were determined using Mothur and

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reference sequences retrieved from the GeneBank database.

19

with 1000 bootstrap repetitions and Jukes-Cantor

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2.4. Morphological characterization of enrichment cultures

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Biomass samples from the SBR were collected and 5 µl aliquots were uniformly spread on a

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glass microscope slide. Phase contrast microscopy was performed on a Nikon i80 microscope

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(Nikon Instruments Inc., NY) at 40x * 10x with image acquisition using Nikon camera and

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SPOT 4.6 Advanced software (Diagnostic Instruments Inc., MI). For obtaining representative

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size distribution of the enriched cultures, 368 objects (cells or cell aggregates) were sized, as

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recommended in a previous study

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(Scion Corporation, MD) to measure a projected two-dimensional cross-sectional area of

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visualized cells or cell aggregates. The projected area diameter (da), which corresponds to the

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diameter of an equivalent circle with the projected cross-sectional area (A) of the cells or cell

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aggregates, was calculated using Eqn. 1 21.

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da =2ට π

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where,

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da: projected area diameter, µm

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A: projected cross-sectional area, µm2.

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2.5. Determination of biokinetic parameters

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Biokinetic parameters describing the Nitrospira spp. enrichment were estimated using extant

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respirometric assays as previously described

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washed three times with nitrogen free feed-medium at pH 7.5. Extant respirometric assays were

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performed in duplicate 100 mL jacketed glass vessels, which were filled with washed biomass

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and sealed with a Clark-type polarographic DO electrode (YSI Model 5300A, Yellow Springs,

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OH). Respirometric assays were initiated by injecting 8.7 µL of a 75 g N/L stock of NaNO2 into

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the respirometric vessels to achieve an initial nitrite concentration of 4 mg-N/L. DO consumption

20

. Images were post-processed on Scion image software

A

(1)

22

. Briefly, biomass withdrawn from the SBR was


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in response to NO2--N oxidation was continuously monitored at 1 Hz in LabVIEW (National

178

Instruments, Austin, TX) using custom-built virtual instruments. The maximum specific growth

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rate (µmax) was obtained based on the maximum oxygen uptake rates measured during a given

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batch respirometric assay (Eqn. S1, Supplementary Information). The oxygen half saturation

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coefficient (KO) was estimated from four batch experiments (Table S1, Supplementary

182

Information) under non-limiting nitrite concentrations. Similarly, the nitrite half saturation

183

coefficient (KS) and biomass yield coefficient (Y) were estimated under non-limiting DO

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concentrations (achieved using air or pure oxygen, Table S1, Supplementary Information).

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Raw respirograms (DO profiles as a function of time) were transformed using the slope function

186

in MS ExcelTM to yield oxygen uptake rate (OUR) profiles over time. DO and OUR profiles

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were processed as shown in Figure S1 and S2 and equations S1 - S4 (Supplementary

188

Information).

189

The thermodynamics and growth stoichiometry of the enriched Nitrospira were expressed in

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terms of the fraction of electrons channeled for biosynthesis (fs) and energy transfer efficiency

191

(ߝ) 23, as shown in equations S5 - S7 (Supplementary Information).

192 193

3. Results and discussion

194

3.1. Reactor performance

195

Over the course of the study, the average maximal degree of nitrite removal at the end of each

196

phase of reactor operation was 99.8 ± 0.2 % (n = 4), 95.8 ± 2.9 % (n = 5), 93.0 ± 2.1 % (n = 2)

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and 97.0 ± 3.7 % (n = 8), respectively (Fig. 1). The corresponding conversion of nitrite to nitrate

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was 95.3 ± 2.2 %, 96.5 ± 10.1 %, 98.8 ± 2.2 % and 97.2 ± 3.9, respectively. Within each

199

operating phase, transient nitrite accumulation was observed in response to step increases in the



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influent nitrite concentrations, followed systematically by near complete oxidation of nitrite to

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nitrate. The purpose of gradual increase of influent nitrite concentrations was to increase the

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amount of enriched Nitrospira spp. Indeed, as the average biomass concentrations in the reactor

203

gradually increased from 161.3 ± 14.6 mg/L (n = 3), 183.5 ± 7.5 mg/L (n = 3), 308.1 ± 7.4 mg/L

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(n = 3) to 361.7 ± 37.3 mg/L (n = 8) in each phase, respectively, effluent nitrite concentrations

205

gradually decreased and the reactor achieved ultimate steady state.

206 207

3.2. Microbial community composition during selective enrichment of Nitrospira spp.

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The initial SBR biomass samples harbored a broad diversity of microorganisms, which included

209

AOB, NOB and heterotrophic bacteria (Fig. 2). Methylotrophic bacteria were also detected

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likely owing to the use of methanol as an electron and carbon source in the full-scale wastewater

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plant, from which the SBR was inoculated. Among AOB and NOB, Nitrosomonas spp. and

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Nitrobacter spp. were initially detected in the SBR biomass (Fig. 2). The concentrations of

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Nitrospira spp., Nitrobacter spp., and AOB during SBR start-up were 7.0 × 107 ± 1.2 × 106, 3.0

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× 107 ± 8.4 × 106 and 1.0 × 106 ± 2.6 × 104 gene copies/mL, respectively (Fig. 3). During the

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course of enrichment, the concentrations of Nitrospira spp. increased to a terminal range of 7.7 ×

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108 ± 7.5 × 107 gene copies/mL towards the end of phase 4, while the concentrations of

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Nitrobacter spp. and AOB decreased to 1.7 × 106 ± 1.2 × 105 and 1.0 × 105 ± 1.7 × 104 gene

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copies/mL, respectively (Fig. 3). The enriched Nitrospira spp. were closely related to Candidatus

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Nitrospira defluvii (97% 16S rRNA sequence similarity), which is affiliated with Nitrospira

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lineage I (Fig. 4). Based on qPCR, Nitrospira spp. contributed to 99.6 ± 0.1 % (n = 12) of the

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total bacteria 16S rRNA gene copy number at the end of phase 4 of enrichment.


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A possible explanation of the high enrichment of Nitrospira spp. could be their reported higher

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affinity for nitrite and DO (KS = 0.1 – 1.1 mg-N/L; KO = 0.5 – 0.6 mg-O2/L) compared to

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Nitrobacter spp. (KS = 0.3 – 7.6 mg-N/L; KO = 0.2 – 4.3 mg-O2/L) 24-29. Indeed, when the reactor

225

was under ultimate steady-state operation, in-situ SBR cycle profiles showed consistently non-

226

detectable nitrite concentrations (Fig. 5).

227

Previous studies have indeed shown that nitrite concentrations are the primary driver for the

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competition between Nitrospira spp. and Nitrobacter spp., with higher nitrite concentrations (80

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– 520 mg-N/L in the bulk liquid) favoring the growth of Nitrobacter spp. over Nitrospira spp.

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and vice-versa

231

influence possible niche differentiation of Nitrospira strains from different phylogenetic lineages

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33, 34

233

lineage II

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concentrations observed each time the influentt nitrite concentrations were stepped up, might

235

have selected for Nitrospira of lineage I rather than lineage II in this study.

236

Beside nitrite concentrations, operational DO concentration was a critical parameter affecting

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Nitrospira community composition in the nitrifying reactors. The SBR DO concentrations in this

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study were in the range of 0.5 -1.1 mg-O2/L, which is close to previously reported KO values (0.5

239

– 0.6 mg-O2/L)

240

previously, long-term exposure to low DO concentrations below 0.5 mg-O2/L resulted in an

241

enrichment of Nitrospira spp. over Nitrobacter spp.16 Furthermore, within Nitrospira

242

themselves, limiting DO concentrations favored Nitrospira lineage I over lineage II 35. In other

243

studies, NOB exhibited a lag phase in nitrate production or nitrite consumption after anoxic

244

periods in continuous reactors resulting in transient nitrite accumulation 36, 37.

1, 4, 15, 30-32

. Furthermore, extant nitrite concentrations have been suggested to

. Nitrospira defluvii (lineage I) displayed higher nitrite tolerance compared to the member of 33, 34

. Based on these previous findings, the transiently non-limiting nitrite

25, 29

and thereby suggests some degree of DO limitation as well. As described


spp.


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From a general perspective, the resulting ecology and kinetics of engineered biological processes

246

are a function of the operating configuration and influent characteristics. Alternative reactor

247

design and operating strategies could possibly result in different organisms and kinetics.

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However, such differentiation was not the main focus of this study.

249

In this study, we demonstrate Nitrospira spp. proliferation at the expense of nearly all other

250

bacteria in a solely nitrite-fed SBR subjected to long-term dual DO and nitrite limitation.

251

Although growth of AOB has been shown at sustained limiting to non-detectable ammonia

252

concentrations

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range 0.33 ± 0.05 % (n = 3), 0.03 ± 0.02 % (n = 3), 0.01 ± 0.01 % (n = 4), and 0.02 ± 0.003 % (n

254

= 4) of the total 16S rRNA gene copies, respectively, during the four phases of enrichment. The

255

possibility of ammonia oxidizing archaea (AOA) proliferation at limiting ammonia

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concentrations as demonstrated previously 41 was not interrogated in the SBR, given the principal

257

focus on NOB herein. Heterotrophic bacteria (belonging to the genera Weeksella, Schlesneria,

258

Methylovorus and Propionivivibrio) were detected despite the absence of organic carbon in the

259

SBR influent, potentially supported by organic metabolic by-products released by the NOB

260

themselves 42.

261

The results of qPCR and 16S rRNA gene amplicon sequencing are expected to be somewhat

262

different. Our aim was to be completely transparent and thus we included both sets of results.

263

Specifically, the difference of Nitrospira spp abundance calculated using qPCR and 16S rRNA

264

gene amplicon sequencing can be attributed to specificities of primers between Nitrospira 16S

265

rRNA gene and universal 16S rRNA gene. For assay validation, specificities of Nitrospira and

266

total bacteria 16S rRNA primer sets were checked using Test-Prime by running in silico PCR on

267

the SILVA databases 43. The specificities for the Nitrospira and total bacteria suite of primers are

38-40

, relatively negligible fractions of AOB were measured in this study, in the


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100% and 56%, respectively, with no mismatches. This means that Nitrospira 16S rRNA gene

269

primer set can cover Nitrospira spp more specifically than universal 16S rRNA gene primer set.

270

Also, qPCR results are expected to be more quantitative because the qPCR data was an absolute

271

quantification based on a standard curve using known concentrations of plasmid.

272

On the other hand, 16S rRNA gene amplicon sequencing is useful for identification and

273

classification of the bacterial community.

274 275

3.3. Morphology of enriched Nitrospira spp cultures.

276

The Nitrospira spp. enrichment was largely comprised of planktonic cells with an overall

277

average characteristic diameter of 1.3 ± 0.6 µm (Fig. 6). Cell aggregates were occasionally

278

observed and were in the size range 3 - 4 µm, significantly lower than those previously reported

279

in activated sludge in the range 73 - 593 µm,

280

nearly devoid of filamentous microorganisms, which contribute symptomatically towards

281

aggregation and floc formation in activated sludge 44, 45.

282

The observed planktonic morphology of the Nitrospira spp. enrichment cultures was distinct

283

than reported in previous studies, where Nitrospira spp., were present in cell aggregates either by

284

themselves or in conjunction with other AOB, NOB and heterotrophic bacteria

285

previously reported that Ca. Nitrospira defluvii secreted increasing degrees of extracellular

286

polymeric substances (EPS) when grown in batch culture with increasing initial nitrite

287

concentrations. The secreted EPS could potentially directly contribute to cell aggregation or

288

support the growth of flocculant heterotrophic biomass

289

Nitrospira defluvii appeared under conditions of long periods of starvation, increasing cell

25, 29

. Furthermore, SBR biomass samples were

34

3, 34, 46, 47

. It was

. In another study, planktonic cells of



14

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density and nitrate accumulation

. However, the underlying basis for the planktonic

291

morphology enriched herein remains to be conclusively determined.

292

Nevertheless, the specific reason for the low degree of cellular aggregation in the Nitrospira spp.

293

biomass enriched herein is not exactly clear and needs further mechanistic investigation.

294 295

3.4. Kinetic characterization of planktonic Nitrospira spp.

296

The average µmax of the Nitrospira spp. enrichment was 0.69 ± 0.10 d-1 (Table 2), which

297

corresponded to an average doubling time (td) of 1 d. The estimated KS value of 0.52 ± 0.14 mg-

298

N/L in Nitrospira spp. (lineage I Ca. Nitrospira defluvii) was lower than the KS values of

299

Nitrobacter spp. reported in previous studies

300

hypothesis that Nitrospira in lineage II have higher affinity for nitrite than these organisms in

301

lineage I

302

Nitrospira lineage I (KS = 0.42 mg-N/L), which was lower than that of Nitrospira lineage II (KS

303

= 0.14 mg-N/L)33. Estimated KO values of 0.33 ± 0.04 mg-O2/L in Nitrospira spp. determined in

304

this study were also lower than those reported for Nitrobacter spp.

305

affinities for genus Nitrospira have been studied previously

306

Nitrospira lineage I (Ca. Nitrospira defluvii) was determined for the first time in this study.

307

Our results are in alignment with the widely held notion that Nitrospira spp. are K-strategists

308

possessing relatively higher affinity for nitrite and oxygen. Further, the low propensity for cell

309

aggregation observed in this study likely resulted in lower substrate mass-transfer limitation and

310

correspondingly lower estimates of the nitrite and oxygen affinity coefficients (KS and KO,

311

respectively), relative to previous studies with a higher degree of aggregation. Overall, such

312

theoretical expectations and experimental observations are also consistent with the relative

33, 49

27, 28, 48

. Moreover, previous studies suggested the

. Indeed, the KS value in this study was close to reported nitrite affinity of

7, 25


26, 29

While the oxygen

, the KO value of planktonic

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enrichment of Nitrospira spp. in mixed culture biofilms, where they can be exposed to substrate

314

limitation in terms of nitrite, DO and even inorganic carbon 1, 31, 32.

315

From a fundamental perspective, Nitrospira spp. typically encode for periplasmic nitrite

316

oxidoreductase (NXR), which is involved in the oxidation of NO2--N to NO3--N 50. In contrast,

317

Nitrobacter spp. encode for a cytoplasmic NXR 51, 52, which requires the transport of NO2--N and

318

NO3--N in opposite directions across the inner membrane. Periplasmic oxidation of NO2--N by

319

Nitrospira spp. has multiple advantages, including the generation of a higher proton motive force

320

(for subsequent energy transduction) per unit NO2--N oxidized

321

needed trans-membrane NO2--N and NO3--N exchange. Together, these factors could potentially

322

contribute to the higher competitive advantage of Nitrospira spp. over other NOB under a wide

323

variety of nitrite limited environments including conventional engineered BNR processes.

324

Thermodynamics and stoichiometry of growth

325

Estimated values of the biomass yield coefficient (Y) of the Ca. Nitrospira defluvii enrichment

326

were 0.14 ± 0.02 mg biomass COD produced/mg-N oxidized, calculated as described previously

327

53

328

oxidized, using an oxygen-nitrite equivalence of 1.14 mg-O2/mg-N (Table 3) and assuming

329

preferential assimilation of NH3 and CO2 for biosynthesis

330

electron efficiency (ߝ) 23 for the Ca. Nitrospira defluvii enrichment herein was 0.7 (Table 3). The

331

estimated values of Y, fS and ε, were within the range computed based on values reported in

332

literature for Nitrospira spp. enrichments (Table 3). Furthermore, descriptors of the growth

333

stoichiometry (Y and fS) and thermodynamic efficiency (ε) for Nitrospira spp. were uniformly

334

higher than for Nitrobacter spp. (Table 3). These results are notable, given that most previous

335

studies focus on biokinetics while characterizing or comparing different types of NOB. In

50

and the lack of otherwise

. This corresponds to a fS value of 0.12 ± 0.02 mg biomass COD produced/mg NO2--NOD

22, 53


. The corresponding computed


336

particular, this is the first study to estimate thermodynamic parameters including both biomass

337

yield coefficient and energy transfer efficiency for Nitrospira lineage I. Rather, the higher

338

electron capture efficiency and biomass yields of Nitrospira spp. also likely contribute to their

339

increased competitiveness in nitrite- (or even oxygen-) limited environments. Based on this

340

study, engineered strategies typically associated with shortcut biological nitrogen removal

341

(BNR) processes such as sustained low DO operation appear to be inappropriate to select against

342

Nitrospira spp. Rather, alternatives such as intermittently non-limiting DO concentrations (as

343

engendered by periodic aeration of sequential aerobic-anoxic zones) could be more appropriate.

344

The results of this study actually then provide the underlying basis for NOB out-selection

345

approaches such as the AOB versus NOB (AVN) control technology 2, 54.

Page 16 of 32

346 347

In sum, we report the selective enrichment of planktonic Nitrospira spp. cultures in a continuous

348

reactor process along with corresponding estimates of key biokinetic, stoichiometric and

349

thermodynamic coefficients. In general, our hypotheses regarding selective enrichment of

350

Nitrospira spp. under sustained NO2--N and DO limitation were largely supported by

351

experimental results. As the wastewater industry transitions to more energy efficient BNR

352

alternates that require NOB out-selection or washout, it becomes necessary to develop a rigorous

353

understanding of the eco-physiology, biokinetics, thermodynamics of both the desirable (in casu,

354

aerobic and anaerobic AOB) and undesirable (in casu, NOB) protagonists. Subsequently, this

355

understanding could potentially be used in conjunction with mechanistic models to develop

356

engineered bioprocess strategies to achieve both clean water as well as energy-efficiency

357

objectives.

358


Page 17 of 32


359

Acknowledgements

360

This study was supported by Water Environment & Reuse Foundation and the Hampton Roads

361

Sanitation District.

362

Supporting Information Available This information is available free of charge via the Internet

363

at http://pubs.acs.org.

364



365

Page 18 of 32

Table 1. Summary of primers information for qPCR. Target gene

Primer

Nucleotide Sequence (5'-3')

Universal

1055F

ATGGCTGTCGTCAGCT

16S rRNA

1392R

ACGGGCGGTGTGTAC

Nitrospira

NTSPAf

CGCAACCCCTGCTTTCAGT

16S rRNA

NTSPAr

CGTTATCCTGGGCAGTCCTT

Nitrobacter

Nitro-1198f

ACCCCTAGCAAATCTCAAAAAACCG

16S rRNA

Nitro-1423r

CTTCACCCCAGTCGCTGACC

amoA-1F

GGGGTTTCTACTGGTGGT

amoA-2R

CCCCTCKGSAAAGCCTTCTTC

amoA

Reference 55

56

57

366 367 368


58

Page 19 of 32

369


Table 2. Summary of biokinetic parameters NOB

Nitrospira spp.

µmax

KS

KO

(1/day)

(mg-N/L)

(mg-O2/L)

0.69 ± 0.10

0.52 ± 0.14

0.33 ± 0.04

This studya

0.9 – 1.1

0.54

29a

0.11 – 0.50

0.47

25b

0.45 - 0.52

28c

0.13 – 0.39

59c

0.18

48b

0.3 – 1.7 1.2 – 1.3

Nitrobacter spp.

0.38 – 1.69

29a

0.17 – 4.32

26b

0.39 – 1.28

0.69 – 7.6

28c

0.48

1.49

27a

0.77 370

a

enriched culture

371

b

mixed culture

372

c

pure culture

Reference

373 374


60b


Page 20 of 32

375

Table 3. Summary of calculated stoichiometric and thermodynamic coefficients describing

376

biomass growth

Culture type

fSb

Y (mg-COD/mg-N)

(mg-COD/mg-

ߝ

Reference

NOD)

enriched Nitrospira

0.14 ± 0.02

0.12 ± 0.02

0.7

This study

cultures

0.12 - 0.2a

0.11 - 0.18

0.64 - 0.86

29

enriched

0.08a

0.07

0.51

24

0.07 – 0.1a

0.06 - 0.09

0.51 - 0.58

27

0.1a

0.62

61

0.1

0.62

62

Nitrobacter cultures NOB in nitrifying enriched cultures NOB

0.12a

377

a

reported values in literature.

378

b

NO2--N has a nitrogenous oxygen demand of 1.14 mg-O2/mg-N.

379 380


Page 21 of 32


381

382 383

Figure 1. Performance of the enrichment SBR. Error bars represent the standard deviation of

384

duplicate measurements.

385 386 387 388 389 390 391 392



Page 22 of 32

393 394

Figure 2. Microbial community composition (at the genus level) during the course of enrichment

395

(phase 1, day 0 – 23; phase 2, day 24 – 57; phase 3, day 58 – 99; phase 4, day 100 – 222),

396

describing the the top sixteen most abundant genera.

397 398 399


Page 23 of 32


400 401

Figure 3. Concentrations of total bacteria, Nitrospira spp., Nitrobacter spp., and AOB in the

402

enrichment SBR. Error bars represent the standard deviation of triplicate measurements.

403 404 405 406 407 408



409 410

Figure 4.

Phylogenetic affiliation among sequences from fifty high-abundance operational

411

taxonomic units after final enrichment (Day 158 sample) based on next generation sequencing of

412

16S rRNA gene amplicons with references in the genus Nitrospira. The tree was constructed

413

using the neighbor-joining algorithm and nodes supported by bootstrap values are indicated.

414

Scale bar represents 1% sequence divergence.

415


Page 24 of 32

Page 25 of 32


416 417

Figure 5. Profiles of SBR nitrite, nitrate and DO concentrations during a representative 6 h cycle

418

on day 222. Error bars represent the standard deviation of triplicate measurements.

419



Page 26 of 32

420 421

Figure 6. Phase contrast microscope image of Nitrospira spp. cells (at 40X * 10X magnification).

422

Samples obtained on day 222 of SBR operation.

423 424


Page 27 of 32


425

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