Experimental Evidence for in Situ Nitric Oxide Production in Anaerobic

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Environmental Processes

Experimental evidence for in situ nitric oxide production in anaerobic ammonia-oxidizing bacterial granules Rathnayake M. L. D. Rathnayake, Mamoru Oshiki, Satoshi Ishii, Takahiro Segawa, Hisashi Satoh, and Satoshi Okabe Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00876 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Environmental Science & Technology

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For submission to Environmental Science & Technology as a Research Article

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Experimental evidence for in situ nitric oxide

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production in anaerobic ammonia-oxidizing bacterial

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granules

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Rathnayake M. L. D. Rathnayakea,b, Mamoru Oshikib,c, Satoshi Ishiib,d,e, Takahiro Segawaf,g,

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Hisashi Satohb*, Satoshi Okabeb

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a

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Peradeniya, 20400, Sri Lanka.

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b

Department of Civil Engineering, Faculty of Engineering, University of Peradeniya,

Division of Environmental Engineering, Graduate School of Engineering, Hokkaido University,

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North-13, West-8, Sapporo 060-8628, Japan.

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c

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Nishikatakaimachi, Nagaoka, Niigata 940-8532, Japan.

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d

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Upper Buford Circle, St. Paul, MN 55108, USA.

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e

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Avenue, St. Paul, MN 55108, USA

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f

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409-3898, Japan

Department of Civil Engineering, National institute of Technology, Nagaoka College, 888

Department of Soil, Water and Climate, University of Minnesota, 439 Borlaug Hall, 1991

BioTechnology Institute, University of Minnesota, 140 Gortner Laboratory, 1479 Gortner

Center for Life Science Research, University of Yamanashi, 1110, Shimokato, Chuo, Yamanashi

ACS Paragon Plus Environment


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g

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Midori-cho, Tachikawa, Tokyo 190-8518, Japan.

Transdisciplinary Research Integration Center, National Institute of Polar Research, 10-3

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Email addresses:

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Rathnayake M. L. D. Rathnayake – [email protected]

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Mamoru Oshiki – [email protected]

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Satoshi Ishii – [email protected]

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Takahiro Segawa – [email protected]

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Hisashi Satoh – [email protected]

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Satoshi Okabe – [email protected]

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Keywords

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Anammox granules; Factors affecting nitric oxide production; In situ analysis; Nitric oxide

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production pathways; Stable isotope-labeling studies with inhibitors.

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ABSTRACT

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Although nitric oxide (NO) emissions from anaerobic ammonium oxidation (anammox)-based

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processes were reported previously, the NO production pathways are poorly understood. Here,

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we investigated the NO production pathways in anammox granules in detail by combining 15N-

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stable isotope tracer experiments with various inhibitors, microsensor measurements, and

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transcriptome analysis for key genes of NO2– reduction. NO was emitted from the anammox

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granules, which account for 0.07% of the N2 emission. 15N-stable isotope-tracer experiments


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indicated that most of the N2 was produced by anammox bacteria, whereas NO was produced

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from NO2– reduction by anammox and denitrifying bacteria. The NO emission rate was highest

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at pH 8.0 and accelerated by increasing NH4+ and NO2– concentrations in the culture media. The

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microsensor analyses showed the in situ NO production rate was highest in the outer layer of the

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anammox granule where anammox activity was also highest. The detected in situ NO

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concentrations of up to 2.7 µM were significantly above physiological thresholds known to affect

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a wide range of microorganisms present in wastewater. Hence, NO likely plays pivotal roles in

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the microbial interactions in anammox granules, which needs to be further investigated.

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INTRODUCTION

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Nitric oxide (NO) is an important atmospheric trace gas1. It has a direct effect on the ozone

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chemistry of the atmosphere2 and it is a free radical3 and toxic to a wide range of organisms4–6.

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NO molecules have ecological influences on microbial consortia by regulating specific microbial

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activities7,8, the metabolisms of nitrifiers9, and the formation10 and dispersion of bacterial

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biofilms11,12.

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NO emissions have been reported in wastewater treatment processes13,14, in which NO

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emission rates accounted for 0.003–11.3% of the nitrogen load of the processes. A key to

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formulating strategies to control and reduce NO emissions from wastewater treatment processes

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is to accumulate knowledge of the mechanisms of NO production and consumption. NO can be

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produced in wastewater treatment processes via the following three biochemical pathways: 1)

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denitrification, in which nitrite (NO2−) is reduced to NO by cytochrome cd1-type or copper-

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containing nitrite reductases15; 2) anaerobic ammonium oxidation (anammox) process, in which



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NO is produced from NO2− reduction and hydroxylamine (NH2OH) oxidation16; and 3)

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nitrification, in which the nitrosyl radical (NOH) can be produced as an intermediate in oxidation

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of NH2OH and the unstable NOH is biologically transformed to NO17. NO can also be converted

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to nitrous oxide (N2O) by NO reductase, which decreases NO emissions from a wastewater

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treatment tank. Various physicochemical parameters, such as concentrations of dissolved

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oxygen13,18, nitrogenous compounds13,19, organic and inorganic carbon, and pH20, affect the

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above metabolic pathways. Thus, the pathways of NO production and consumption are complex

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and yet to be fully resolved.

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Application of anammox granular sludge to wastewater treatment has received increasing

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attention because a granular sludge process has several advantages over dispersed sludge, such as

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good settling characteristics, high biomass retention in the reactor, and the ability to withstand

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shocks and toxic loadings21–24. However, NO emission from anammox granules has been

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reported20,23,25,26. Efforts should be made to determine the pathways of NO production and

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consumption to prevent NO emission from anammox granules. Although a few experimental

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studies have revealed the mechanisms of NO production from anammox granules20,25, the

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pathways of NO production inside the granules have not been investigated probably because it is

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difficult to monitor NO turnover inside anammox granules: NO can be produced and consumed

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at the microscale within a single granule by several different types of the metabolic reactions

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described above27–29.

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In this study, experimental evidence for in situ NO production and consumption within

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single anammox granules taken from a lab-scale anammox reactor was accumulated by a

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combination of batch experiments with 15N-stable isotope-labeled substrates, inhibitors,

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microsensor measurements, dye staining of NO, and transcriptome analysis. Furthermore,


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physicochemical parameters affecting the NO emissions were investigated. The batch

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experiments with inhibitors were carried out to distinguish the contribution of anammox and

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denitrification processes to NO production. Based on the results obtained, the pathways of NO

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production in anammox granules are discussed.

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MATERIALS AND METHODS

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Anammox granules

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A lab-scale up-flow column reactor (Bio column reactor, Fujisaki, Osaka, Japan) was operated at

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37°C in the dark to cultivate anammox granules. The reactor volume was 220 mL and seeding

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biomass (anammox granules with 3–5 mm diameter) collected from another anammox reactor29

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was inoculated with a 70% (v/v) packing ratio. The column reactor received effluent of a partial

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nitrification (PN) reactor30 continuously with 1.1 h of hydraulic retention time. The column

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reactor configurations were previously described30.

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Activity tests

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Standard anaerobic techniques were employed in an anaerobic chamber (Coy Laboratory

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Products, Grass Lake Charter Township, MI, USA) where oxygen concentration was maintained

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at 99.99995%) by vacuuming (2 min) and purging (1 min). 15N-labeled NO2–

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purchased from Cambridge Isotope Laboratories (Andover, MA, USA) and non-labeled NH4+

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were added to the vials at final concentrations of 5 mM and the biomass was incubated for 12 h

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at 37°C in the dark.

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Contributions of anammox and denitrification to 31NO production were investigated by

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supplementation with 1 mM methanol33 and 500 mg L−1 penicillin G34, respectively (Supporting

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Information (SI) Figure S1), and by increasing the NO2− concentration from 2.5 to 20 mM as

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shown in Table 1. A vial containing autoclaved biomass was prepared as a negative control.

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The influences of NH4+ and NO2− concentrations and pH on specific activities of 31NO

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production were examined over the range 2 to 30 mM non-labeled NH4+ and 15N-labeled NO2−

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and over the pH range 6.0 to 9.0. The pH was adjusted by adding 1 M H2SO4 or 1 M NaOH.

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Microsensor measurements of NH4+, NO2–, NO3–, NO and N2O concentrations and pH

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Ion-type NH4+, NO2–, NO3–, and pH microsensors were fabricated and calibrated as previously

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described30. N2O and NO-specific microsensors were purchased from Unisense (Arhus,

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Denmark) and calibrated according to the instructions provided by the manufacturer. The steady-

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state concentration profiles of NH4+, NO2–, NO3–, NO, and N2O in anammox granules, as well as

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the pH, were measured as previously described30. Before the measurements, anammox granules

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were acclimated in 2.5 L of inorganic nutrient medium (4.5 mM NH4+ and NO2–) in anoxic

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conditions for at least 3 h to ensure that steady-state profiles were obtained. Physicochemical


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parameters of the medium were almost unchanged during the microsensor measurements. The

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measurement using the NO microsensor was repeated with supplementation with 10 µM 2-(4-

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carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a scavenger of

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NO molecules, to examine the specificity of the NO microsensor. Net volumetric emission rates

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of NO, N2O, NH4+ and NO2– were estimated from the measured concentration profiles30.

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Diffusion coefficients at 25°C of 1.38×10–5, 1.25×10–5, 2.10×10–5 and 2.21×10–5 cm2 s–1 for

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NH4+, NO2–, N2O and NO, respectively, were used to calculate the net volumetric production

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rates30,35.

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Imaging of NO accumulation in anammox granules by DAF-FM DA-dye staining

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Spatial accumulation of NO molecules in anammox granules was investigated by staining the

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granules with 4-amino-5-methylamino-2’,7’-difluorescein diacetate (DAF-FM DA). DAF-FM

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DA is a cell-membrane permeable dye that is hydrolyzed intracellularly to a cell-membrane

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impermeable form, DAF-FM. DAF-FM specifically reacts with NO molecules and emits green

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fluorescence36; therefore, intracellular NO accumulation can be examined by fluorescence

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microscopy36. Anammox granules were anoxically incubated in the inorganic nutrient medium

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containing 4.5 mM NH4+ and NO2– at 37°C in the dark. After 3 h of preincubation, DAF-FM DA

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(Sekisui Medical, Tokyo, Japan) was supplemented at a final concentration of 10 µM, and the

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granules were incubated for a further 30 min. Incubation without NH4+ and NO2– was also

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conducted as a negative control for DAF-FM DA staining. The stained granules were washed

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three times with sterile phosphate buffer solution, embedded into Tissue-Tec OCT (optimum

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cutting temperature) compound (Sakura Finetek, Torrance, CA), and then sliced into 10-µm-

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thick specimens with a cryostat CM 1510S (Leica, Bensheim, Germany). The specimens were



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placed on a gelatin-coated microscope slide (Cellline Associates) and dried in the dark. Green

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fluorescence of DAF-FM was observed using a model LSM510 confocal laser-scanning

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microscope (Carl Zeiss, Oberkochen, Germany).

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Transcriptome analysis

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Comparative transcriptome analysis was performed to identify genes active under NO-producing

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conditions. Anammox granules incubated in conditions I–IV (Table 1) were collected from the

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vials at the end of the incubation and stored in RNA later solution (Thermo Fisher Scientific) at

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−80°C to prevent degradation of RNA molecules. Total RNA was extracted using TRIzol RNA

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Isolation Reagent (Life Technologies) and a Direct-zol RNA Miniprep Kit (Zymo Research), as

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described in the manufacturer’s protocol. Absence of DNA contamination in the extracted total

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RNA was confirmed by PCR targeting anammox bacterial 16S rRNA genes37. Ribosomal RNA

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in the total RNA was removed using a Ribo-Zero rRNA Removal Kit (Bacteria) (Epicenture)

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according to the manufacturer’s protocol. The resulting mRNA samples were used to prepare

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sequencing libraries, which was done using the NEB Next Ultra RNA Library Prep Kit for

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Illumina (New England Biolabs). Briefly, mRNA was fragmented by heating at 85°C for 5 min;

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then, first and second strands of the complementary DNA (cDNA) were synthesized. After end-

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repair and adaptor ligation, cDNA fragments were enriched by PCR for 14 cycles. In this PCR

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step, sample-specific index sequences were also attached to the fragments. PCR amplicons (200–

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800 bp) were recovered and purified after agarose gel electrophoresis by using the GeneRead

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Size Selection Kit (Qiagen). Concentrations of the enriched cDNA fragments were measured

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using a BioAnalyzer 2100 (Agilent). Equal amounts of cDNA fragments from the four samples


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were mixed and used for MiSeq sequencing (Illumina). Paired-end sequencing reaction was

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performed using the MiSeq Reagent Kit v.2 (250 bp × 2).

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Sequence reads were mapped against the “Candidatus Brocadia sinica” genome

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(GenBank accession number BAFN01000001 through BAFN01000003)38 using Bowtie 239. The

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values of fragments per kilobase per million mapped reads (FKMF) were counted and

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normalized by gene length using the HTSeq-count program40. BLASTn analysis was also carried

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out to identify sequence reads of functional genes involved in nitrification and denitrification that

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were not located in the “Ca. Brocadia sinica” genome. The sequence reads were subjected to

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BLASTn analysis (cutoff e-value and hit length 100 bp, respectively) against narG

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(encoding respiratory nitrate reductase), nirS, nirK, norB, nosZ (nitrous oxide reductase) and

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amoA (ammonia monooxygenase) retrieved from the FunGene database41. Computations of the

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BLASTn analysis were carried out using the National Institute of Genetics (NIG) supercomputer

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(https://sc.ddbj.nig.ac.jp/index.php). Relative abundance of the hit sequences was calculated

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using the equation:

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Relative abundance (%) = number of the hit sequences/number of total sequence reads.

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All statistical analyses, including principal component analysis (PCA), were performed in the

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statistical computing software R.

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Chemical and microbiological analyses

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NH4+, NO2– and NO3– concentrations were determined by ion chromatography, as described

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previously30. The total nitrogen (NH4+, NO2– and NO3–) removal efficiency (T-NRE) was

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calculated by dividing the total nitrogen removal rate (T-NRR) by the total nitrogen loading rate

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(T-NLR). Concentrations of 15N-labeled gaseous compounds (i.e., 29N2, 30N2, 30NO and 31NO)



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were determined by gas chromatography-mass spectrometry (GC-MS) analysis as described

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previously31. Biomass concentration was determined as protein concentration using bovine

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serum albumin (BSA) as a standard, as described previously31. Protein concentration was

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determined with a DC Protein Assay kit (Bio-Rad). Specific activities of 29N2 and 31NO

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production were calculated based on the changes in gas concentrations during the initial active

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gas emission phase of the batch test (20 mM NH4+ were about twice those at

Experimental Evidence for in Situ Nitric Oxide Production in Anaerobic

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