Expression of the nirS, hzsA, and hdh Genes in Response to Nitrite

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Expression of the nirS, hzsA, and hdh Genes in Response to Nitrite Shock and Recovery in Candidatus Kuenenia stuttgartiensis Yayi Wang, xiao Ma, Shuai Zhou, Ximao Lin, Bin Ma, Hee-Deung Park, and Yuan Yan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00546 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 28, 2016

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Environmental Science & Technology

Expression of the nirS, hzsA, and hdh Genes in Response to Nitrite Shock and

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Recovery in Candidatus Kuenenia stuttgartiensis

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Yayi Wang* , Xiao Ma , Shuai Zhou , Ximao Lin , Bin Ma , Hee-Deung Park , Yuan

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5 6 7 8 9

10 11

†

†

†

†

‡

§

†

Yan

†

State Key Laboratory of Pollution Control and Resources Reuse, College of

Environmental Science and Engineering, Tongji University, Siping Road, Shanghai 200092, P. R. China ‡

Key Laboratory of Beijing for Water Quality Science and Water Environment

Recovery Engineering, Engineering Research Center of Beijing, Beijing University of

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Technology, Beijing 100124, P. R. China

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Anam-Dong, Seongbuk-Gu, Seoul 136-713, South Korea

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ABSTRACT: In this study, Candidatus Kuenenia stuttgartiensis were subjected to

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of mRNA levels of cytochrome cd 1 nitrite:nitric oxide oxidoreductase (nirS),

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Changes in the hydrazine dehydrogenase (HDH) protein level were monitored. At 200

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(nSAA) slightly increased relative to the control despite a significant decrease in nirS,

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increased nirS, hzsA, and hdh mRNA levels were observed, but the nSAA decreased,

13 15

§

School of Civil, Environmental and Architectural Engineering, Korea University,

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distinct nitrite shocks (66 (control), 200, 300, 400 and 500 mg N/L), and the responses

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hydrazine synthase (hzsA), and hydrazine dehydrogenase (hdh) were assessed.

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mg NO 2 --N/L, the normalized specific anaerobic ammonium-oxidizing activity

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hzsA, and hdh mRNA levels. When nitrite increased to 300 and 400 mg N/L,

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relative to the 200 mg NO 2 --N/L exposure. HDH protein detection revealed that 1

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Candidatus Kuenenia stuttgartiensis attempted to yield high enzyme levels by

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shock, the nirS, hzsA, and hdh mRNA levels decreased, alongside decreased nSAA

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our findings advance understanding of the mechanisms that anammox bacteria use to

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improve the diagnostic accuracy of bioreactor failures when nitrite accumulation

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stimulating mRNA synthesis to resist the nitrite-induced stress. On 500 mg NO 2 --N/L

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and HDH levels. Although the mRNA levels did not always coincide with activities,

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cope with nitrite inhibition at the transcriptional and translational levels, which will

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occurs.

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Table of Contents

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ANAMMOXOSOME NO2

-

(

)

(300 mgN/L

→400 mgN/L)

Protein activity (

)

Protein level (

)

Increased protein level

HDH

66 200 300 400 500

Nitrite concentration (mg N/L)

DNA Transcription

mRNA (

)

Translation

Increased mRNA level

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2


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INTRODUCTION

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biological nitrogen removal from wastewater around the world,1 owing to lower

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compared to conventional nitrification and denitrification processes. A hypothetical

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nitrite to nitric oxide is catalyzed by cytochrome cd 1 nitrite:nitric oxide

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combining ammonium with nitric oxide, and finally hydrazine is oxidized to

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Anaerobic ammonium oxidation (anammox) is increasingly important in

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sludge production, no need for organic carbon, and substantial energy savings as

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metabolic pathway of anammox bacteria has been proposed: first, the reduction of

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oxidoreductase (NirS), then hydrazine synthase (HZS) produces hydrazine by

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dinitrogen gas (N 2 ) by hydrazine dehydrogenase (HDH).2

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including nitrite, 3, 4 even though it is an essential substrate of these bacteria.5 The

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rate may result in the failure of anammox processes after nitrite shock, which has been

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technology in wastewater treatment. A wide range of nitrite inhibition thresholds (70–

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difficult to judge the reversible or irreversible nature of nitrite inhibition and to design

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Anammox bacteria are highly sensitive to various inhibiting compounds,

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sensitivity of anammox bacteria to nitrite toxicity combined with their slow growth

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regarded as one of the main bottlenecks constraining the application of anammox

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2000 mg N/L) have been reported for anammox bacteria (Table S1),4-9 which makes it

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anammox-based processes for wastewater treatment.

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determining specific substrate (e.g., ammonium and nitrite) removal rates.4-10

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ammonia-oxidizing bacteria (AOB) and denitrifying bacteria may compete for similar

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anammox bacteria, nitrite-oxidizing bacteria and denitrifying bacteria compete for

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The influence of nitrite on anammox activity has mostly been studied by

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However, in anammox bioreactor systems where many bacterial species coexist,

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substrates. For example, anammox bacteria and AOB compete for ammonium, and

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nitrite, which leads to inaccurate measurements of ammonium and nitrite used by

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highly sensitive and specific method to estimate the effect of nitrite inhibition on

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anammox bacteria. Further research is urgently required to develop an accurate,

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anammox activity.

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conditions,11-13 mRNA-based technologies have been used to monitor the biomass

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organisms (PAOs)12 in varying environments (Table S2). Most studies showed that

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example, the nitrite reduction in Candidatus Brocadia fulgida (anammox bacteria)

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with the decreased hydrazine oxidoreductase (hzo) mRNA levels.13 However, other

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activities.15, 16, 21, 22 Cua and Stein16 examined expression of the key genes of AOB

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toxicity. They found that in the presence of 280 mg NO 2 --N/L, the decreased amoA

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Nitrosospira multiformis, and the unchanged levels of amoA mRNA of Nitrosomonas

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suggested that, even comparing closely related species, each strain has its own

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correlations between biomass activities and mRNA transcription levels highlight that

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compounds, which should be explored independently.

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Because of the quick responses of mRNA levels to changes in environmental

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activities of AOB,14-16 denitrifying bacteria,17 and polyphosphorus-accumulating

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biomass activities positively correlate with the associated mRNA levels.12, 17-20 For

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ceased after 2 h inhibition of 450 mg NO 2 --N/L and aeration, which was consistent

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studies demonstrated that mRNA levels were not linked directly with biomass

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including Nitrosomonas eutropha and Nitrosospira multiformis in response to nitrite

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mRNA levels did not lead to a signiﬁcant decrease in the nitrite production rate in

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eutropha did not correlate with the decreased nitrite production rate. Their findings

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adaptive and regulatory mechanisms to cope with nitrite shock. The uncertain

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organisms might have different mechanisms to cope with stress from inhibiting

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Although gene transcription in anammox bacteria in response to nitrite shock (hzo) 4


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and starvation conditions (hzsA) has been evaluated in a few studies,13, 23 very few

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and hdh at the transcription and translation levels on exposure to nitrite shock.

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activity has not been well investigated.

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have comprehensively assessed responses of the key functional genes of nirS, hzsA,

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Moreover, the correlation between the abundance of mRNA transcripts and anammox

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In this study, transcription of genes encoding components of the principal

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metabolic pathway, including those for cytochrome cd 1 nitrite: nitric oxide

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quantitatively measured in Candidatus Kuenenia stuttgartiensis using reverse

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was employed to specifically quantify the protein level of HDH, as the hdh gene is

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of Candidatus Kuenenia stuttgartiensis (anammox bacteria) to different nitrite

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effect of nitrite shock on the anammox performance and recovery of Candidatus

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levels and HDH protein level to different nitrite concentrations in short-term exposure

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mRNA transcript levels of the nirS, hzsA, and hdh genes in Candidatus Kuenenia

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nitrite concentration shocks.

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MATERIALS AND METHODS

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anammox sequencing batch reactor (SBR) with a working volume of 20 L (in a water

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oxidoreductase (nirS), hydrazine synthesis (hzsA) and hydrazine oxidation (hdh), was

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transcriptase quantitative polymerase chain reactions (RT-qPCR). Western blotting

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one of the most diagnostic phylogenetic markers for anammox bacteria.2 The response

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loadings was assessed. The main objectives of this study were to: (1) determine the

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Kuenenia stuttgartiensis; (2) identify the response of the nirS, hzsA, and hdh mRNA

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experiments and recovery batch tests; and (3) explore the possibility of employing the

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stuttgartiensis as a physiological indicator for the anammox activity under different

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Parent Anammox Reactor Setup and Operation. A sealed laboratory-scale

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bath) was operated at 33 ± 1°C. One standard SBR cycle was 8 h, consisting of a

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10-min effluent decanting period, and a 15-min idle phase. In the 10-min feeding

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reaction period, constant mixing was achieved with a mechanical stirrer with a speed

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for 270 days, the mixed liquid volatile suspended (VSS) concentration was

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approximately 75% and 25%, respectively; the system achieved a stable volumetric

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in situ hybridization (FISH) using the hybridization probes EUB338 mix and

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Candidatus Kuenenia stuttgartiensis, which made up 81 ± 9% of the total microbial

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Information (SI) text 1.

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(g/L): NH 4 Cl, 1.07 (280 mg NH 4 +-N/L); NaNO 2 , 1.77 (360 mg NO 2 --N/L); KHCO 3 ,

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Na 2 EDTA, 0.02. A trace element solution was also added to 0.5 mL/L. The trace

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CuSO 4 ·5H 2 O, 0.625; MnCl2 ·4H 2 O, 0.495; NaMoO 4 ·2H 2 O, 0.55; CoCl2 ·6H 2 O, 0.6;

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10-min filling period, a 390-min anoxic period, a 55-min sludge settling period, a

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phase, 10 L of synthetic wastewater was pumped into the reactor. In the anoxic

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of 80 rpm. The influent pH was adjusted to 7.0 by adding 2 M HCl. After cultivation

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approximately 5 g/L, with granules and flocculent sludge accounting for

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substrate (ammonium and nitrite) removal rate of about 0.99 kg N/m3·d. Fluorescence

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KST-1275 showed that the main specie of anammox bacterium in the reactor was

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community. The detailed procedures of FISH assays are presented in Supporting

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Synthetic Wastewater. The synthetic wastewater used in this study contained

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1.25; KH 2 PO 4 , 0.02; CaCl2 , 0.02; MgSO 4 ·7H 2 O; 0.08; FeSO 4 ·7H 2 O, 0.015; and

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element solution contained (g/L): EDTA, 7.5; H 3 BO 3 , 0.035; ZnSO 4 ·7H 2 O, 1.075;

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and NiCl2 ·6H 2 O, 0.475.

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nitrogen transformations and the response of the mRNA transcription and protein

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Nitrite Exposure and Recovery Batch Test Procedure. To evaluate the

levels of Candidatus Kuenenia stuttgartiensis to increasing nitrite concentrations, 6


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batch experiments were performed with a fixed initial ammonium concentration of 50

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N/L. For recovery tests, the sludge was washed with a washing medium (the same

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nitrite exposure tests, and then anammox reactions proceeded with, initially, 50 mg

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All batch experiments were conducted in plexiglass reactors with a 0.5-L

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mg N/L, and initial nitrite concentrations of 66 (control), 200, 300, 400, and 500 mg

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composition as the synthetic wastewater but without NH 4 Cl and NaNO 2 ) after the

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N/L ammonium and 66 mg N/L nitrite.

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reaction volume, which were placed in a water bath with a magnetic stirrer to ensure a

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to monitor pH variations; during the batch experiments, the pH was maintained at 7.5

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originated from the parent SBR at the end of the anoxic reaction after stable anammox

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washing medium. The biomass concentration was 5 g VSS/L at the beginning of batch

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10 min to remove dissolved oxygen. The reactor was sealed with gas-tight rubber

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constant temperature (33 ± 1°C) and mixing. Online pH probes were used in reactors

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± 0.1 by adding 0.1 M HCl or 0.1 M NaOH. The anammox biomass for these tests

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performance was achieved. Before use, the sludge was washed and resuspended in the

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tests. The gas and liquid phases were purged with an Ar/CO 2 (95/5%) gas mixture for

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stoppers and anaerobically stirred for 5 h.

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decrease of the substrate concentration during the 5-h experiment and related to the

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with respect to the SAA of the control test (normalized SAA (nSAA, %) =

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The SAA was calculated from the maximum slope of the curve described by the

biomass concentration in the reactor.10 The SAA of each batch test was normalized

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(SAAinhibited /SAA control ) × 100).

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and hdh were selected as the functional genes for inferring anammox activity. Sludge

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DNA Isolation, RNA Extraction, cDNA Synthesis and RT-qPCR. nirS, hzsA,

samples were regularly collected along with measurements of the consumption of 7



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ammonium. DNA was extracted using the DNAzol reagent (Invitrogen, Shanghai,

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Total RNA was isolated using the TRIzol reagent (Invitrogen) and purified with a

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presented in Supporting Information (SI) text 2. The quality and quantity of DNA and

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Scientific, Shanghai, China). Reverse transcription was conducted with the

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RT-qPCR amplification was performed using an ABI 7500 system (Applied

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(Takara). The standard curves for RT-qPCR were generated through serial decimal

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are summarized in Table S3.

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anammox bacterial 16S rDNA concentrations. For comparison of the mRNA

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mRNA/DNA data corresponding to the time when ammonium was close to depletion

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normalized with respect to the value in the control test. The levels of DNA and mRNA

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significance of the results and p < 0.05 was considered statistically significant.

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B-PER® Bacterial Protein Extraction Reagent (Pierce, Shanghai, China), and the

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antibody against HDH protein in Candidatus Kuenenia stuttgartiensis was produced

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China) and purified with a universal DNA purification kit (Tiangen, Shanghai, China).

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RNA clean kit (Tiangen). The detailed extraction methods of DNA and RNA are

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RNA were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher

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PrimeScript® RT kit (Perfect Real Time) with gDNA Eraser (Takara, Shanghai, China).

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Biosystems, Shanghai, China) with SYBR® Premix Ex Taq™ (Tli RNaseH Plus)

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dilutions of plasmid DNA carrying the specific target gene inserts. All the primer pairs

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The mRNA concentrations of nirS, hzsA, and hdh were normalized to the

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abundances among the different nitrite exposure and recovery assays, the

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in each batch test was selected, and each mRNA/DNA ratio in batch tests was

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were measured in duplicate. Analysis of variance (ANOVA) was used to test the

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Antibody Production and Western Blotting. Total protein was extracted using

protein concentration was determined with a BCA Protein Assay kit (Pierce). The

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by Invitrogen. Western blotting was conducted following the procedures described by

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Pinck et al.24

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Millipore filter units (0.45-μm pore size) for the analysis of NH 4 +-N and NO 2 --N.

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methods.25 Hydrazine concentration was detected using the method described by Watt

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Other Analytical Methods. Liquid samples were immediately filtered through

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NH 4 +-N, NO 2 --N, and VSS were measured in accordance with standard

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and Chrisp.26

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RESULTS

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mRNA Levels in One Cycle in the Control Reactor. As it is only around 20 years

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is incomplete, especially at the molecular level. Figure 1 presents the changes in the

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cycle of the control reactor. Ammonium and nitrite were consumed almost

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ammonium and nitrite were 0.18 ± 0.02 g N/g VSS·d and 0.26 ± 0.03 g N/g VSS·d,

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1:1.39 ± 0.02, similar to the expected theoretical value of 1:1.32, suggesting a typical

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The transcription levels of nirS, hzsA, and hdh mRNA followed similar trends to

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Nitrogen Transformations and the Changes in the nirS, hzsA, and hdh

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since the first report of anammox, knowledge of the metabolism of anammox bacteria

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nirS, hzsA, and hdh mRNA levels and the transformations of nitrogen in a typical

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simultaneously in the control batch (Figure 1; Table 2). The nitrogen removal rates of

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respectively, and the stoichiometry of ammonium conversion to nitrite removal was

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anammox reaction occurred in the control test.27

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each other in the control assay (Figure 1). After ammonium and nitrite were added

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amount of nirS, hzsA, and hdh mRNA responded rapidly, increasing approximately

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into the reactor at initial concentrations of 50 mg N/L and 66 mg N/L respectively, the

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10-, 6-, and 6-fold, respectively, at 135 min, compared with those at the starting point 9



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of the control test. These data suggest that the substrates were a strong inducing factor

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pathway. The anammox reaction (i.e., a decrease in both the ammonium and nitrite

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These results were in good agreement with previous studies on AOB and denitrifying

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Nitrosomonas eutropha at 6 h was 15-fold higher than that at the starting point of 600

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activity of Pseudomonas mandelii (a denitrifying bacterium) and observed that in the

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compared with that at the starting point, whereas a 225-fold increase was observed

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indicate that transcription levels of functional genes involved in catabolic pathways

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for the transcription of genes (nirS, hzsA and hdh) coding for the principal metabolic

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concentrations) corresponded with the increased mRNA levels (Figure 1; Table 2).

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bacteria. Aoi et al.22 found that the amoA mRNA level of Nitrosomonas europaea and

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mg N/L ammonium incubation. Saleh-Lakha et al.17 examined the denitrification

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absence of KNO 3 treatment, the nirS gene expression increased only 12-fold

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when the KNO 3 level was 1000 mg N/L. These analogous trends in gene expression

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can be significantly upregulated when substrates become available.

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decreased because of the exhaustion of ammonium and nitrite (Figure 1), confirming

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During the latter 165 min of the reaction, the three mRNA levels significantly

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that the half-life values for the bacterial mRNAs are short.28

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Concentrations Shocks. As Figure 2a and b shows, although influent ammonium

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mg NO 2 --N/L led to obvious differences in the anammox reaction rate and the

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removal rate and the nSAA were 0.21 ± 0.03 g N/g VSS·d and 117.2 ± 11.3%,

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± 0.02 g N/g VSS·d and 100 ± 12.7%, respectively) (p < 0.05).

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The nirS, hzsA, and hdh mRNA Responses to Different Nitrite

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was consumed in all tests, exposure to concentrations of 66, 200, 300, 400, and 500

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transcription of nirS, hzsA, and hdh. On 200 mg NO 2 --N/L exposure, the ammonium

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respectively (Table 1), which were slightly higher than those in the control assay (0.18

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However, when the concentrations of nitrite increased to 300, 400, and 500 mg

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N/L, the nSAA decreased to 95.6 ± 3.1%, 81.1 ± 1.6%, and 52.1 ± 3.1% (p < 0.05),

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concentrations of 225.3 ± 20.8, 333.5 ± 17.7 and 438.0 ± 1.4 mg N/L, respectively

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concentrations. The 50% activity inhibition for nitrite (IC 50 ) was approximately 500

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anammox bacteria reported by Lotti et al.4 and Carvajal-Arroyo et al.,9 respectively.

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substrate-utilizing activities,13, 23 and thus mRNA may be used as a biomarker for

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bacterial species form complex consortia.22 However, in the present study, the amount

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and 7 ± 1.8% (p < 0.05) of their levels in the control assay, respectively, in response to

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nSAA at this nitrite concentration (Figure 2b). When compared with those observed in

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and 400 mg NO 2 --N/L resulted in elevated nirS, hzsA, and hdh mRNA levels (p
200 mg NO 2 --N/L) had irreversible inhibitory effects on

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agree with those of Scaglione et al., 29 who reported that anammox biomass recovered

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respectively (Figure 3a and b), relative to the control. These results indicated that the

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nitrogen removal given a short recovery time of only one cycle. Our findings partially

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60–80% of its activity after 100, 200, 300, and 500 mg N/L nitrite shocks.

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nirS, hzsA, and hdh mRNA levels recovered to 71.4 ± 2.9%, 63.6 ± 1.7%, and 87.6 ±

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± 3.3%, 114.8 ± 0.7% and 87.8 ± 1.7%, respectively, of their levels in the control

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recovery test after 400 mg N/L nitrite exposure, where higher recovery of the nirS,

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shock, reaching values of 130.5 ± 8.5%, 115.3 ± 0.6%, and 138.6 ± 0.9% (p < 0.05),

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value (Figure 3b). The findings from the recovery test after 400 mg N/L nitrite

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and anammox activity.

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stuttgartiensis to Identify the Nitrite Inhibition Mechanism at the Translational

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catalytic functions of the NirS, HZS, and HDH proteins. Any factor associated with

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of the anammox reaction. We proposed two plausible explanations for our observed

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After short-term exposure to 200, 300 and 500 mg N/L nitrite, respectively, the

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2.3%, respectively, 91.4 ± 0.3%, 72.3 ± 0.5% and 88.0 ± 1.5%, respectively, and 87.6

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assay (p < 0.05) (Figure 3b). However, particular attention should be paid to the

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hzsA, and hdh mRNA levels was observed than after 200, 300 and 500 mg N/L nitrite

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respectively, of the control levels, but the nSAA only recovered to 87% of the control

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exposure further demonstrated an inconsistent relationship between mRNA abundance

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Western Blotting Detection of HDH Protein in Candidatus Kuenenia

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Level. Whether or not the anammox reaction can happen ultimately depends on the

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the synthesis and function of NirS, HZS, and HDH may influence the overall outcome

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inconsistency between mRNA levels and nSAA in Candidatus Kuenenia 13



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stuttgartiensis: 1) the gene transcription process (from DNA to mRNA) or the gene

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resulting in insufficient protein levels; or 2) the activities of the already synthesized or

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nitrite shocks, even if the protein levels remained unchanged or increased.

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the shock, western blotting was used for the detection of HDH protein. It was find that

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4a) when compared with the control assay, demonstrating a type of cellular stress

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increased from 66 to 200 and 300 mg N/L, an overall decrease in the amount of HDH

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the HDH protein level was observed in comparison with that in the 300 mg NO 2 --N/L

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lowest level of HDH protein was detected compared with the other NO 2 --N/L

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hdh mRNA level (Figure 2b).

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to each other after exposure to 66, 200, 300, and 500 mg N/L nitrite (Figure 4b).

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exposure (Figure 4b).

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DISCUSSION

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Candidatus Kuenenia stuttgartiensis to Nitrite Shocks. In the present study, the

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translation process (from mRNA to protein) was retarded by nitrite shock inhibition,

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freshly synthesized NirS, HZS, and HDH were inhibited by higher-concentration

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To verify whether gene translation levels were affected by nitrite shock and after

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nitrite exposure of 200-500 mg N/L caused downregulation of HDH protein (Figure

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response induced by nitrite toxicity. Specifically, when the nitrite shock concentration

330

was observed (Figure 4a). However, at 400 mg N/L nitrite, a significant increase in

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exposure assay (Figure 4a). When the nitrite concentration reached 500 mg N/L, the

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exposure assays (Figure 4a), which corresponded with the lowest observed nSAA and

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In the activity recovery batch tests, the detected HDH protein levels were similar

338 340 342

However, a high level of HDH protein was observed after 400 mg N/L nitrite

Insight into the Response of the nirS, hzsA, and hdh mRNA Levels in

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nirS, hzsA, and hdh mRNA abundance sometimes positively coincided with the

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a relationship did not occur at all levels of nitrite exposure, e.g., to 200, 300 and 400

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On 200 mg N/L nitrite shock, the decreased HDH protein level (Figure 4a)

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anammox activity, e.g., in the 66 and 500 mg N/L nitrite exposure scenarios; but such

347

mg N/L nitrite.

349

corresponded to the reduced hdh mRNA level (Figure 2b), indicating that the

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enzymes were stimulated by this level of nitrite, leading to 16% higher observed

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be because there was higher ‘potential’ anammox activity when measured at a

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severely inhibited by the residual nitrite of 138.5 ± 2.1 mg N/L (Table 2)). Similarly,

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amoA mRNA levels did not result in a signiﬁcant decrease in the nitrite production

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reductions in the mRNA levels of amoA and hydroxylamine oxidoreductase (hao) in

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uptake rate. These results indicate that decreased mRNA levels do not always lead to

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post-translational regulation, or variances in the availability of energy, electrons and

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transcription process was inhibited; nevertheless, the overall activities of the reacting

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nSAA relative to the control (Figure 2b). Such an increased anammox activity might

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nonlimiting substrate (nitrite) concentration (assuming that anammox bacteria are not

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in the presence of 280 mg NO 2 --N/L, Cua and Stein (2011)16 found that decreased

358

rate in Nitrosospira multiformis, and Yu and Chandran (2010)15 found that the

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Nitrosococcus mobilis did not correlate with the relatively unchanged specific oxygen

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reduction in biomass activities; other factors, such as post-transcriptional regulation,

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reductant, may also influence the overall biomass activities.30

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hdh increased compared with those in the 200 mg NO 2 --N/L exposure assay (Figure

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the transcription process and the translation process were influenced. In contrast, on

365 367

On exposure to 300 mg N/L nitrite, although the mRNA levels of nirS, hzsA, and

2b), a decreased HDH protein level was detected (Figure 4a), which means that both

15



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exposure to 400 mg N/L nitrite, a remarkable increase in both the hdh mRNA and the

371

(Figures 2b and 4a). We conjecture that Candidatus Kuenenia stuttgartiensis

373

inhibition) by increasing the production of energy-harvesting-related mRNAs (e.g., of

375

enzymes, such as HDH (Figure 4a); however, the activities of both the already

377

nitrite toxicity, resulting overall in a 19% lower observed nSAA compared with that in

379

be an inhibitor of the enzymes of various microorganisms.31-33 Baumann et al.32 found

381

accumulations in the medium. Also, Hinze and Holzer 33 observed that nitrite can

383

glycolysis and gluconeogenesis), and thus spoiled the glycolysis and glycogen

385

increased production of mRNA occurred when the bacteria were exposed to inhibiting

387

exposed to 10, 30, and 90 μM Zn2+, the AMO activity was inhibited by Zn2+, and the

389

respectively; however, a significant increase in the amoA level was only observed

391

replacing Cu2+ in the active site of AMO protein and that Nitrosomonas europaea

393

de novo synthesis of key enzymes, such as AMO protein. For this purpose, the amoA

370

HDH protein levels was observed relative to those on 300 mg N/L nitrite exposure

372

attempted to compensate for the reduced enzyme activities (caused by nitrite-induced

374

nirS, hzsA, and hdh) (Figure 2b), which led to increased production of the key related

376

synthesized and freshly synthesized HDH protein may have been inhibited by in situ

378

the control test (Figure 2b). Indeed, nitrite at a high concentration has been reported to

380

that synthesized nitrite reductase was inactivated by nitrite, resulted in nitrite

382

react with and inactivate glyceraldehyde-3-phosphate dehydrogenase (involved in

384

synthesis processes. A similar regulatory mechanism was observed in AOB, where

386

compounds.11, 14 Radniecki et al.14 found that when Nitrosomonas europaea was

388

ammonium-dependent specific oxygen uptake rate decreased by 12%, 92%, and 94%,

390

after the addition of 30 and 90 μM Zn2+. They hypothesized that Zn2+ was irreversibly

392

attempted to overcome the zinc-mediated damage by the generation of new cells and

16


Page 16 of 32

Page 17 of 32


394

mRNA content increased, which could be used to produce new AMO protein. Their

396

levels in 400 mg N/L nitrite exposure assays in the present study; nevertheless, the

398

When increasing the nitrite concentration to 500 mg N/L, the reduction in the

395

explanation could help explain the inconsistency of the nSAA and the HDH protein

397

exact mechanism involved requires further study.

399

levels of mRNA transcription and HDH protein (Figures 2b and 4a) could be

401

the requirements of synthesis of nirS, hzsA, and hdh mRNA and HDH protein,

403

activities. The lower HDH protein level and nSAA on 500 mg N/L nitrite exposure

405

study, also suggested that the intracellular inhibitory mechanisms and responses were

407

and hdh were lower in both of these tests than in the control test (Figure 2b).

409

Candidatus Kuenenia stuttgartiensis at 200–400 mg nitrite/L is likely a stress response

411

inconsistency between the biomass activities and mRNA levels was observed; after

413

levels in Candidatus Kuenenia stuttgartiensis could be a failure to resist high nitrite

415

abundance positively coincided with the anammox activity. The findings of the

417

potential regulatory mechanisms at the transcriptional and translational levels on

400

attributed to the failure of Candidatus Kuenenia stuttgartiensis to provide energy for

402

because the anammox reaction was retarded owing to seriously inhibited enzyme

404

compared with that on 200 mg N/L nitrite exposure (Figures 2b and 4a), shown in this

406

completely different in these two cases, even though the mRNA levels of nirS, hzsA

408

Taken together, the increment in the nirS, hzsA, and hdh mRNA levels in

410

to detoxify nitrite when the bacteria are subject to nitrite inhibition, during which

412

exposure to nitrite at 500 mg N/L, the decrease in the nirS, hzsA, and hdh mRNA

414

concentration stress; in this case, we found that the nirS, hzsA, and hdh mRNA

416

present study advance our understanding of anammox bacterial stress responses and

418

exposure to high nitrite concentration. In addition, these outcomes may improve the 17



419

diagnostic accuracy of anammox bioreactor failures when nitrite inhibition occurs.

421

to each other after exposure to 66, 200, 300, and 500 mg N/L nitrite (Figure 4b).

423

exposure (Figure 4b). We hypothesize that inhibited HDH protein was present in the

425

shock (Figure 4). In that case, Candidatus Kuenenia stuttgartiensis have to synthesize

427

compensate for the decreased enzyme activity. Thus, higher total HDH protein (a

429

after 400 mg NO 2 --N/L shock compared with the control assay (Figures 3b and 4b).

431

mRNA levels were produced during the activity recovery batch tests after 500 mg

433

(Figures 3b and 4b), relative to those in the 500 mg N/L nitrite exposure assay (Figure

420

In the activity recovery batch tests, the detected HDH protein levels were similar

422

However, a high level of HDH protein was observed after 400 mg N/L nitrite

424

Candidatus Kuenenia stuttgartiensis as an inactive enzyme after 400 mg NO 2 --N/L

426

higher levels of hdh mRNA (Figure 3b) for synthesis of fresh, active HDH protein, to

428

combination of inhibited and fresh HDH protein) and mRNA levels were detected

430

Similarly, due to the serious decreased activity on 500 mg N/L nitrite exposure, higher

432

NO 2 --N/L shock (Figure 3b), which led to the increased HDH protein level and nSAA

434

2b).

436

to address the transcriptional response of the nirS, hzsA, and hdh genes of the

438

Our findings provide strong evidence that an increase in the abundance of nirS, hzsA,

440

different nitrite exposure conditions. More importantly, we highlight that the mRNA

442

concentrations and enzyme activities (e.g., HDH) during high-nitrite exposure, which

435

Significance of this Study and Prospects for the Future. This study is the first

437

Candidatus Kuenenia stuttgartiensis group to different nitrite concentration exposures.

439

and hdh mRNA does not necessarily indicate an increase in anammox activity in the

441

levels were simultaneously affected by various factors including substrate (i.e., nitrite)

443

should be considered when analyzing mRNA data from engineered or environmental 18


Page 18 of 32

Page 19 of 32


444

samples.

446

the regulatory mechanisms in anammox bacteria at the mRNA and protein levels in

448

genetic properties of anammox bacteria. Understanding the mechanisms that

450

translational levels could lead to more sophisticated and reliable anammox process

452

digester centrate, where nitrite accumulation may frequently occur.

454

concentration exposures highlight the need to understand the specific regulatory

456

factors in both wastewater treatment plants and natural systems. In the present study,

458

to different nitrite concentrations and in recovery tests; the HDH activity, as well as

460

studied.

462

ASSOCIATED CONTENT

464

This file contains additional methods details and Tables S1–S4. This material is

445

These findings fill a knowledge gap between observed anammox activities and

447

high-nitrite environments, improving our understanding of the physiological and

449

anammox bacteria use to cope with nitrite inhibition at the transcriptional and

451

control when treating high-ammonia-content wastewater, such as that from anaerobic

453

The distinct responses of anammox bacteria we observed to different nitrite

455

mechanisms employed by anammox bacteria to counter nitrite and other stressful

457

among proteins, we only explored the HDH levels in anammox bacteria on exposure

459

the variations in the other two key proteins (NirS and HZS), need to be further

461 463

Supporting Information Available

465

available free of charge via the Internet at http://pubs.acs.org.

467

AUTHOR INFORMATION

466 468

*Corresponding Author 19



469

Tel: +21 65984275; E-mail: [email protected];

471

Shanghai 200092, P. R. China

473

ACKNOWLEDGMENTS

475

(NSFC) (nos. 51078283 and 51178325). The Fundamental Research Funds for the

477

Key Laboratory of Pollution Control and Resource Reuse (Tongji University), China

479

assistance with RT-qPCR studies.

470 472

College of Environmental Science and Engineering, Tongji University, Siping Road,

474

This work was supported by the National Natural Science Foundation of China

476

Central University (Tongji University) (0400219238), and the Foundation of State

478

(PCRRY 0400231010), are also acknowledged. We thank Dr. Hong Wang for

20


Page 20 of 32

Page 21 of 32


480

REFERENCES

482

O.; Haaijer, S.; van der Star, W.; Schmid, M.; van de Vossenberg, J.; Schmidt, I.; Harhangi, H.;

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H.; Harhangi, H. R.; Janssen-Megens, E. M.; Francoijs, K. J.; Stunnenberg, H. G.; Keltjens, J. T.;

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van Loosdrecht, M.; Kuenen, J. G.; den Camp, H. O.; Strous, M., 1994-2004: 10 years of research

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Jetten, M. S. M.; Strous, M., Molecular mechanism of anaerobic ammonium oxidation. Nature

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(7) Fux, C.; Marchesi, V.; Brunner, I.; Siegrist, H., Anaerobic ammonium oxidation of

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(6) Dapena-Mora, A.; Fernandez, I.; Campos, J. L.; Mosquera-Corral, A.; Mendez, R.; Jetten, M. S.

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nitrogen gas production. Enzyme. Microb. Tech. 2007, 40 (4), 859–865.

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ammonium-rich waste streams in fixed-bed reactors. Water Sci. Technol. 2004, 49 (11-12), 77-82.

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Pre-exposure to nitrite in the absence of ammonium strongly inhibits anammox. Water Res. 2014,

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(9) Jung, J.; Kang, S.; Chung, Y.; Ahn, D., Factors affecting the activity of anammox bacteria

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(10) Tang, C. J.; Zheng, P.; Mahmood, Q.; Chen, J. W., Start-up and inhibition analysis of the

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Anammox process seeded with anaerobic granular sludge. J. Ind. Microbiol. Biot 2009, 36 (8),

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Bioeng. 2008, 99 (5), 1085–1095.

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dynamic EBPR conditions. Isme Journal 2011, 5, (2) 329-340.

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Anaerobic Digestion Reject Water. Environ. Sci. Technol. 2010, 44 (16), 6110–6116.

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Continuously Cultured Nitrosomonas europaea Cells Exposed to Zinc Chloride Additions.

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(15) Yu, R.; Chandran, K., Strategies of Nitrosomonas europaea 19718 to counter low dissolved

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Denitrification Activity in Pseudomonas mandelii. Appl. Environ. Microbiol. 2009, 75 (15), 5082–

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Environ. Microbiol. 2005, 71 (3), 1276–1282.

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(20) Beyer, S.; Gilch, S.; Meyer, O.; Schmidt, I., Transcription of Genes Coding for Metabolic

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Molecular Microbiology and Biotechnology 2009, 16 (3-4), 187–197.

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and their amoA mRNA levels in activated sludge by real-time PCR. Water Sci. Technol. 2004, 50

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Profiles, Gene Expression and N-Removal in Anammox Bioreactors Treating Municipal

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(14) Radniecki, T. S.; Semprini, L.; Dolan, M. E., Expression of merA, amoA and hao in

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Biotechnol. Bioeng. 2009, 102 (2), 546–553.

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oxygen and high nitrite concentrations. Bmc Microbiology 2010, 10 (1), 70.

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in three ammonia-oxidizing bacteria. Fems Microbiol. Lett. 2011, 319 (2), 169-175.

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Trevors, J. T., Effect of Nitrate and Acetylene on nirS, cnorB, and nosZ Expression and

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potential ammonia-oxidizing activity and amoA mRNA levels of Nitrosospira briensis. Appl.

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bacteria. J. Ind. Microbiol. Biot 2010, 37 (7), 751–757.

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Key Functions in Nitrosomonas europaea during Aerobic and Anaerobic Growth. Journal of

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as a new biomarker of ammonia oxidation activities in a complex microbial community. Lett. Appl.

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(23) Ali, M.; Oshiki, M.; Okabe, S., Simple, rapid and effective preservation and reactivation of

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of ammonia oxidizers of the beta-subclass of the class Proteobacteria. Appl. Environ. Microbiol.

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Microbiol. 2004, 39 (6), 477–482.

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anaerobic ammonium oxidizing bacterium "Candidatus Brocadia sinica". Water Res. 2014, 57,

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(24) Pinck, C.; Coeur, C.; Potier, P.; Bock, E., Polyclonal antibodies recognizing the AmoB protein

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Public Health Association: Washington, DC, 1998.

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Chem. 1952, 24, (12), 2006-2008.(27) Strous, M.; Heijnen, J.; Kuenen, J.; Jetten, M., The

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(26) Watt, G. W.; Chrisp, J. D., Spectrophotometric method for determination of hydrazine. Anal.

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sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic

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Microbiol. Biot. 1998, 50 (5), 589-596.

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nitrite concentrations of anammox biomass from two SBR fed on synthetic wastewater and landfill

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(30) Kuo, H.-W. Population (amoA-based) and activity (amoA-mRNA-based) assessment of

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Dissertation, University of Tennessee, Knoxville, 2006

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acid (FNA) in wastewater treatment plants. Water Res. 2011, 45 (15), 4672–4682.

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ammonium-oxidizing microorganisms. Appl. Microbiol. Biot. 1998, 50 (5), 589–596.

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tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl.

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(28) Belasco, J. G. mRNA degradation in prokaryotic cells: an overview; Academic Press, New

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leachate. Chem. Eng. J. 2012, 209, 62–68.

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ammonia oxidizing bacteria (AOB) during activated sludge wastewater treatment. Ph.D.

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(31) Zhou, Y.; Oehmen, A.; Lim, M.; Vadivelu, V.; Ng, W. J., The role of nitrite and free nitrous

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(32) Baumann, B.; van der Meer, J. R.; Snozzi, M.; Zehnder, A. J., Inhibition of denitrification

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activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH.

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(33) Hinze, H.; Holzer, H., Analysis of the energy metabolism after incubation of Saccharomyces

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Antonie van Leeuwenhoek 1997, 72 (3), 183–189.

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cerevisiae with sulfite or nitrite. Arch. Microbiol. 1986, 145 (1), 27–31.

572

24


Page 24 of 32

Page 25 of 32

573 574 575


Table 1 Nitrite removal rate, ammonium removal rate, specific anammox activity, and hydrazine concentration in the shock and recovery phases Nitrite concentration

Nitrite removal rate

Ammonium removal rate

Specific anammox activity

Hydrazine concentration

mg N/L

g N/g VSS·d

g N/g VSS·d

g N/g VSS·d

g N/g VSS·d

SP

RP

SP

RP

SP

RP

66

0.26 ± 0.03

0.21 ± 0.05

0.18 ± 0.02

0.15 ± 0.04

0.44 ± 0.05

0.35 ± 0.08

200

0.29 ± 0.05

0.21 ± 0.05

0.21 ± 0.03

0.15 ± 0.03

0.49 ± 0.08

0.36 ± 0.08

300

0.25 ± 0.03

0.19 ± 0.05

0.17 ± 0.02

0.13 ± 0.03

0.42 ± 0.03

0.33 ± 0.08

400

0.20 ± 0.03

0.17 ± 0.04

0.15 ± 0.01

0.12 ± 0.02

0.35 ± 0.03

0.30 ± 0.06

500

0.11 ± 0.04

0.15v± 0.03

0.09 ± 0.01

0.11 ± 0.01

0.23 ± 0.02

0.26 ± 0.05

SP = Shock phase; RP = Recovery phase; B.D.L = Below the detection limit. 576 577 578 579 580 581 582

25


SP

RP

B.D.L (in all tests)


583 584

Page 26 of 32

Table 2 Variations in nitrite concentration during nitrite exposure batch tests Time

Nitrite concentration (mg N/L)

(min)

a

66a

200a

300a

400a

500a

0

67.6 ± 0.9

205.7 ± 7.5

297.5 ± 17.7

393.0 ± 15.6

500.0± 17.0

30

56.1 ± 0.6

192.0 ± 8.5

284.8 ± 18.6

384.5 ± 13.4

492.5 ± 13.4

60

40.8 ± 0.5

176.6 ± 9.0

272.6 ± 23.2

371.0 ± 17.0

484.0 ± 11.3

90

23.3 ± 0.2

154.5 ± 2.1

260.0 ± 25.5

363.0 ± 19.8

474.5 ± 16.3

135

4.1 ± 1.2b

138.5 ± 2.1b

236.5 ± 33.2

349.0 ± 22.6

465.5 ± 12.0

180

0

133.0 ± 2.8

225.3 ± 20.8b

333.5 ± 17.7b

455.0 ± 7.0

240

0

131.5 ± 2.1

223.0 ± 18.4

324.0 ± 11.3

438.0 ± 1.4b

300

0

130.5 ± 2.1

220.5 ± 16.3

320.0 ± 7.0

429.5 ± 10.6

The initial nitrite concentration in each batch test. The residual concentration of NO 2 --N after NH 4 +-N was depleted.

b

585 586 587 588 589 590

26


Page 27 of 32

Figure 1

12

nirS

hzsA

hdh

NO2--N

NH4+-N

60

10

50

8

40

6 30

4

20

2

10

0 0 593 594 595

70 NH4+-N, NO2--N (mg/L)

Relative mRNA abundance

591 592


50

100

150

200

250

0 300

Time (min)

Figure 1. Nitrogen transformations and the changes in nirS, hzsA, and hdh mRNA

596

levels in one cycle in the control reactor. The influent ammonia and nitrite

598

concentrations of nirS, hzsA, and hdh were normalized to the anammox bacterial 16S

600

respect to that at the starting point of the control test (i.e., the relative nirS, hzsA, and

597

concentrations were 50 mg N/L and 66 mg N/L, respectively. The mRNA

599

rDNA concentration, and each mRNA/DNA ratio in batch tests was normalized with

601

hdh mRNA abundances at 0 min = 1).

27



602

Page 28 of 32

Figure 2 60

(a)

66 mg NO2--N/L 200 mg NO2--N/L

NH4+-N (mg/L)

50

300 mg NO2--N/L

40

400 mg NO2--N/L 500 mg NO2--N/L

30 20 10 0

Relative mRNA abundance, nSAA (% of control)

0

50

100

(b)

120

150 200 Time (min)

250

300

nirS hzsA hdh nSAA

y = -0.234x + 167.55 R2 = 0.9621

100 80 60 40 20 0

The mRNA levels of nirS (×104), hzsA, hdh (×102) (copies/copies)

66

603 604

200 300 400 Nitrite concentration (mg N/L)

500

10

(c) 8

66 mg N/L batch

6 4

nirS hzsA hdh

400 mg N/L batch

300 mg N/L batch

500 mg N/L batch

200 mg N/L batch

2 0 0.20

0.25

0.30 0.35 0.40 SAA (g N/g VSS•d)

0.45

0.50

Figure 2. Impact of nitrite concentration on the variations in (a) ammonium

605

concentration (the initial ammonium concentration was 50 mg N/L); (b) the nSAA

607

slightly increased nSAA in Candidatus Kuenenia stuttgartiensis on 200 mg NO 2 --N/L

606

and the relative mRNA levels of nirS, hzsA, and hdh (the dotted line represents the

608

exposure, and the solid line is a fitted straight-line which shows the 28


Page 29 of 32


609

continual trend of decreased nSAA at higher nitrite); and (c) the correlation between

611

(Candidatus Kuenenia stuttgartiensis accounted for 81 ± 9% of the total biomass by

613

mRNA concentrations of nirS, hzsA, and hdh were normalized to the anammox

615

ratio in batch tests corresponding to the time when ammonium was close to depletion

610

nSAA and the mRNA levels of nirS, hzsA, and hdh in cultures of anammox biomass

612

FISH analysis). Normalized SAA (nSAA, %) = (SAA inhibited /SAA control ) × 100. The

614

bacterial 16S rDNA concentrations. Relative mRNA abundance: each mRNA/DNA

616

was normalized with respect to that in the control test.

617

29



618

Page 30 of 32

Figure 3 60

(a)

NH4+-N (mg/L)

50

66 mg NO2--N/L 200 mg NO2--N/L 300 mg NO2--N/L

40

400 mg NO2--N/L 500 mg NO2--N/L

30 20 10 0 0

50

100

150 200 Time (min)

250

300

Relative mRNA abundance, nSAA (% of control)

160 140

(b)

nirS hzsA hdh nSAA

y = -0.0812x + 118.01

120

2

R = 0.9887

100 80 60 40 20 0

The mRNA levels of nirS (×104), hzsA, hdh (×102) (copies/copies)

66

8

200 300 400 Nitrite concentration (mg N/L)

(c) 500 mg N/L batch

400 mg N/L batch

500

nirS hzsA hdh 66 mg N/L batch

6 300 mg N/L batch

4 200mg N/L batch

2 0 0.26

0.28

0.30 0.32 0.34 SAA (g N/g VSS•d)

0.36

619

Figure 3. Variations in (a) the ammonium concentration (the initial ammonium

621

hzsA, and hdh (the solid line is a fitted straight-line showing the

620

concentration was 50 mg N/L), (b) the nSAA and the relative mRNA levels of nirS,

622

continual trend of decreased nSAA), and (c) the correlation between nSAA and the 30


Page 31 of 32


623

mRNA levels of nirS, hzsA, and hdh in cultures of anammox biomass treated with

625

SAA (nSAA, %) = (SAAinhibited /SAA control ) × 100. The mRNA concentrations of nirS,

627

Relative mRNA abundance: each mRNA/DNA ratio in activity recovery batch tests

629

with respect to that in the control test.

624

different concentrations of nitrite in activity recovery batch experiments. Normalized

626

hzsA, and hdh were normalized to the anammox bacterial 16S rDNA concentrations.

628

corresponding to the time when ammonium was close to depletion was normalized

31



630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648

Figure 4

a)

b)

1

2

3

4

5

1

2

3

4

5

Figure 4. Variations of HDH protein level determined by western blotting (a) in

649

nitrite exposure tests (lane 1, 66 mg nitrite-N/L; lane 2, 200 nitrite-N/L; lane 3, 300

651

activity recovery batch tests after nitrite exposure (lane 1, 66 mg nitrite-N/L; lane 2,

653

500 mg nitrite-N/L). The protein level can be assessed based on the band densities.

650

mg nitrite-N/L; lane 4, 400 mg nitrite-N/L; lane 5, 500 mg nitrite-N/L) and (b) in

652

200 mg nitrite-N/L; lane 3, 300 mg nitrite-N/L; lane 4, 400 mg nitrite-N/L; lane 5,

32


Page 32 of 32

Expression of the nirS, hzsA, and hdh Genes in Response to Nitrite

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