Multivariate and Multiscale Approaches for Interpreting the

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Environmental Processes

Multivariate and multi-scale approaches for interpreting the mechanisms of nitrous oxide emission during pig manure–wheat straw aerobic composting Jinyi Ge, Guangqun Huang, Junbao Li, Xiaoxi Sun, and Lujia Han Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02958 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Environmental Science & Technology

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Multivariate and multi-scale approaches for interpreting the mechanisms of nitrous oxide

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emission during pig manure–wheat straw aerobic composting

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Jinyi Ge, Guangqun Huang, Junbao Li, Xiaoxi Sun, Lujia Han*

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Biomass Resources and Utilization Laboratory, College of Engineering, China Agricultural

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University, Beijing 100083, China

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* Corresponding author.

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China Agricultural University (East Campus), Box 191, Beijing 100083, China

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Fax: 86-10-6273-6778

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Tel: 86-10-6273-6313

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E-mail: [email protected]

1 Environment ACS Paragon Plus


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ABSTRACT: Nitrous oxide (N2O) emission during composting causes nitrogen loss and air

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pollution. The interpretation of N2O emission mechanisms will help to customize composting

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strategies that mitigates climate change. At pile and particle scales, this study characterized N2O

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emission-related variables (gases, ions, and microbes) and their correlations during pig manure–

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wheat straw aerobic composting. Pile-scale results showed that N2O emission mainly occurred in

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mesophilic, thermophilic, and cooling phases; the nitrification by ammonia-oxidizing bacteria

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(AOB) and nitrite-oxidizing bacteria (NOB) coexisted with the denitrification by denitrificans

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(DEN); the major NOB and DEN were Nitrobacter (NOB_Nba) and Thiobacillus denitrificans

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(DEN_Tb), respectively. The mechanisms of nitrification, nitrifier denitrification, and anaerobic

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denitrification in composting particles were initially visualized by confocal laser scanning

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microscopy: Betaproteobacteria (AOB_Beta) sporadically distributed on the outer area of the

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particles, NOB_Nba internally attached to AOB_Beta, and Nitrosomonas europea/Nitrosomonas

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eutropha (AOB_eu) and DEN_Tb concentrated in the interior. Correlation analysis of the

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variables showed that the distribution area of AOB_eu was proportional to N2O emission (R2 =

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0.84); AOB not only participated in nitrification but also nitrifier denitrification; N2O formation

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was mainly from nitrifier denitrification by AOB_eu during the mesophilic–thermophilic phase

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and from denitrification by AOB_eu and DEN during the cooling phase.

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KEYWORDS: aerobic composting, nitrous oxide emission mechanism, multi-scale,

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multivariate


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TOC Art



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INTRODUCTION

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The latest World Meteorological Organization (WMO) Greenhouse Gas Bulletin reports

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that the globally averaged mole fraction of nitrous oxide (N2O) reached a new high of 328.9 ±

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0.1 ppb in 2016.1 As a significant source of greenhouse gas emissions, N2O has a global

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warming potential 265 times that of an equal mass of carbon dioxide (CO2), and it can be

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transported to the stratosphere, which will cause ozone depletion. Accumulative N2O emission

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during composting is an important concern, as it could account for 10% of the total nitrogen

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content of nitrogen-rich composting materials such as manure,2,3 which not only exacerbates air

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pollution but also results in nitrogen loss in fertilizer.4,5 Therefore, an understanding of the

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mechanisms of N2O emission during composting is critical to provide theoretical guidance for

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reducing the environmental impact of N2O.

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Based on the pile scale, previous research analyzed N2O emission-related variables (gases,

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ions, and microbes) and established preliminary mechanisms of N2O emission during

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composting. The results showed that there were two pathways of N2O emission, i.e., aerobic

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nitrification and anaerobic denitrification.5-7 During aerobic nitrification, ammonia-oxidizing

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bacteria (AOB) and/or ammonia-oxidizing archaea (AOA) oxidize ammonium (NH4+) to nitrite

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(NO2–); subsequently, nitrite-oxidizing bacteria (NOB) oxidize NO2– to nitrate (NO3–); as an

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intermediate product of ammonia oxidation, hydroxylamine (NH2OH) is oxidized to N2O.8-10

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During denitrification, NO2– is reduced by denitrificans (DEN) anaerobically, where N2O is an

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intermediate.11 However, according to the research in the area of wastewater treatment, nitrifiers

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such as Nitrosomonas europea/Nitrosomonas eutropha (AOB_eu) belonging to the AOB group

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could also execute denitrification and generate N2O, which is called nitrifier denitrification.12-14

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Does nitrifier denitrification occur during aerobic composting and contribute to N2O emission?


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This question needs to be further addressed.15

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The above research based on the pile scale established a theoretical foundation for

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interpreting the mechanisms of N2O emission during composting. However, these studies

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assuming completely aerobic conditions cannot be used to analyze N2O generated from

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anaerobic denitrification.16,17 The availability of oxygen (O2) is a determining factor in N2O

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production. Manure composts have been considered a microaerobic environment featuring a high

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mineral nitrogen availability and a high concentration of organic carbon resource, which would

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provide favourable conditions for N2O production.18 The aerobic layer–anaerobic core structure

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of composting particles could be a starting point for illumination of the coupling of aerobic

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nitrification, anaerobic denitrification, and N2O emission during composting.19-22 Based on

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Fourier transform infrared microspectroscopy, Ge et al.23 determined the dynamic distribution of

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lipids and aromatics in the composting particles and characterized the aerobic layer–anaerobic

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core structure of the composting particles. Furthermore, Wang et al.24 examined the depth

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profiles of the concentration of dissolved O2 and NO3– in manure particles using microelectrodes.

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The results supported the finding concerning the aerobic layer–anaerobic core structure of

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manure particles and showed a positive correlation between the concentration of NO3– and

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dissolved O2. However, as composting is a biochemical process, the distribution of the chemical

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compounds alone cannot reveal the microbial reactions involved in N2O emission. Fortunately, a

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method for micro-zone analysis, fluorescent in situ hybridization–confocal laser scanning

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microscopy (FISH–CLSM), could provide reference for studying the N2O emission-related

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microbial reactions in the composting particles.25,26

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The aims of this study were to interpret the mechanisms of N2O emission during pig

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manure–wheat straw aerobic composting by characterizing the multiple variables at pile and



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particle scales and their correlations, including quantification of gases [O2, ammonia (NH3), and

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N2O], ions (NH2OH, NH4+, NO2–, and NO3–), and microbes (AOB, AOA, NOB, and DEN) at the

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pile scale and visualization of the microbial distribution (AOB, NOB, and DEN) at the particle

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scale. The research could be useful to reduce nitrogen loss during composting and customize

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composting strategies that mitigate greenhouse gas emissions.

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MATERIALS AND METHODS

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Aerobic composting experiments. The schematic of the aerobic composting reactor, the

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details of composting experiments, and the methods for determining basic physicochemical

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parameters can be found in previous studies.27-29 Pig manure (10.0 kg), wheat straw (1.0 kg), and

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deionized water (1.0 kg) were raw materials and mixed thoroughly by hand. The mixture was

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divided equally into two composting reactors (height 0.40 m, diameter 0.25 m, and effective

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volume 16.00 L) and noted as experiments A and B. The duration of the experiments was 16

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days. Based on the results of previous research,27 intermittent aeration (1 h on/1 h off) with a rate

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of 0.35 L min–1 was employed.

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The sampling was performed on days 0, 2, 4, 6, 8, 12, and 16. Moisture content and organic

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matter content of the samples were determined by standard Test Methods for the Examination of

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Composting and Compost (TMECC).30 Total carbon content and total nitrogen content were

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tested with an elemental analyzer (Vario EL CHNOS, Elementar Analysensysteme GmbH,

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Germany) to calculate the carbon to nitrogen ratio. Each measurement was performed in

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triplicate, and the mean values were presented as the results. The results of the physicochemical

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parameters of the initial composting mixtures are presented in Table 1. The values of the

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moisture content, the organic matter content, and the carbon to nitrogen ratio met the

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requirements of composting facilities.6 As the parameters except for the organic matter content


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showed no significant difference between experiments A and B, the related data of the

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experiments were averaged for the purposes of the rest of the analysis.

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Table 1. Physicochemical parameters of the initial composting mixtures. Physicochemical parameter

Experiment A

Experiment B

p

Moisture content (%)

56.17 ± 2.59

56.47 ± 2.84

0.897

Organic matter content (%, DM) 83.25 ± 1.35

79.25 ± 1.21

0.019

Total carbon (%, DM)

42.02 ± 0.15

43.87 ± 2.19

0.216

Total nitrogen (%, DM)

2.55 ± 0.21

2.78 ± 0.09

0.160

Carbon to nitrogen ratio

16.56 ± 1.41

15.80 ± 1.00

0.489

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Note: values are presented as the mean ± standard deviation determined from three replicates; p,

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significance level; p < 0.05 indicates significant difference between experiments A and B; DM,

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dry weight basis.

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Measurement of temperature, O2 concentration, NH3 emission, and N2O emission at

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the pile scale. The composting temperature and ambient temperature were monitored daily using

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thermocouples (Pt100, Omega Engineering Inc., CT, USA) located in the middle of the

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composting mixtures and the surrounding atmosphere, respectively. Data from the

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thermocouples were documented using a programmed data acquisition system (DT85; DataTaker

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Pty Co., Ltd., Australia). The O2 concentration (volume fraction) in the upper part of the reactors

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was measured using an O2 sensor (O2S-FR-T2-18X, Apollo Electronics Co., Ltd., China).

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Simultaneously, gaseous samples in the upper part of the reactors were collected in 1-L gas bags.

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The NH3 concentration in the bags was immediately analyzed by gas detector tubes (GASTEC,

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Japan). The N2O concentration in the bags was immediately analyzed by gas chromatography



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using a greenhouse gas analyzer (GC-2014; Shimadzu, Japan). Each measurement was

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performed in triplicate, and the results were presented as the mean values.

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Measurement of NH2OH, NH4+, NO2–, and NO3– at the pile scale. A spectrophotometric

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method was verified by Frear et al. for determining the NH2OH concentration in soybean

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leaves.31 However, in the case of crude samples such as composting particles, specific extraction

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is demanded. The extraction of composting samples was conducted according to the TMECC

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standard.30 Briefly, the composting samples were diluted by deionized water (1:10) and the

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solutions were shaken for 2 h at room temperature; the solutions were centrifuged at 9000 × g

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(4°C, 15 min) and then filtered by 0.45-µm Millipore filter papers. The NH2OH concentration of

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the filtrates was then quantified by a spectrophotometer (UV-2550, Shimadzu, Japan) according

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to the study of Frear et al.31 The concentration of NH4+, NO2–, and NO3– of the filtrates was

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determined by a flow injection analyzer (FIAstar5000, Foss, Denmark). The unit was µmol N g–1.

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Each measurement was performed in triplicate, and the results were presented as the mean values.

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Real-time quantitative polymerase chain reaction (qPCR) analysis of the bacterial

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abundance of AOB, AOA, NOB, and DEN at the pile scale. According to previous research,

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the main AOB present during composting was Betaproteobacteria (AOB_Beta);8 the main NOB

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included Nitrobacter (NOB_Nba) and Nitrospira (NOB_Nsp);10,32-34 the main DEN included

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Azoarcus-Thauera

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Thiobacillus denitrificans (DEN_Tb);11,35 the key functional genes of denitrification included

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nitrite reductase (nirS + nirK) and nitrous oxide reductase (nosZ).6 The details of the qPCR

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analysis and the primers used for AOB_Beta, NOB_Nba, NOB_Nsp, DEN_AT, DEN_P, DEN_Tb,

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nirS + nirK, and nosZ are listed in Section 1 and Table S1 in Supporting Information. Each

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qPCR measurement was performed in triplicate, and the results were presented as the mean

denitrificans

(DEN_AT),

Paracoccus


denitrificans

(DEN_P),

and

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values. The qPCR results showed that there was no AOA, NOB_Nsp, DEN_AT, or DEN_P during

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pig manure–wheat straw composting.

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FISH–CLSM analysis of AOB_Beta, AOB_eu, NOB_Nba, and DEN_Tb at the particle

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scale. The preparation of particle samples for FISH–CLSM analysis is detailed in Section 2 in

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Supporting Information. In preliminary experiments, we embedded the particles in an optimal

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cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and tried to section them

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into 10-, 20-, and 50-µm slices with a CM 3050-S cryostat (Leica Microsystems GmbH, Wetzlar,

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Germany). Sectioning was technically difficult due to the fragile structure of the particles. Some

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previous FISH–CLSM research of sludge flocs and mangrove roots did not adopt sectioning.36-38

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Szilveszter et al.37 indicated that the CLSM images of sludge flocs without sectioning were

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similar to those after being sectioned (5 µm), and fluorescent probes penetrated well into the

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uncut flocs. The reason might be that, unlike biological tissues that are relatively dense and

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constitute diffusion barriers hindering the penetration of probes, the loose structure of sludge

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flocs would allow a better penetration, even though they were not sectioned. These results

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implied that sectioning might not be mandatory for the FISH–CLSM analysis of delicate samples.

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Therefore, sectioning was not applied to the composting particles in this study.

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The thickness of the particles lying on the slides was examined by a micro-CT system

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(Skyscan 1275, Bruker microCT, Belgium). A total of 20 particles were investigated, and the

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thickness was measured to be 86.3 ± 24.1 µm (Supporting Information, Figure S1). Given that

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the average diameter of manure particles was 506 ± 5 µm,28 the thickness of the particles lying

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on the slides was much lower than the average diameter. This suggests that the particles may

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have been shaved, in effect due to the steps of the preparation process, such as centrifugation and

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rinse, and then as a result presented a cross section that gave us access to the interior of the



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particles.

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The qPCR results showed that there was no AOA, NOB_Nsp, DEN_AT, or DEN_P in the

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composting samples. Therefore, the research objects in FISH–CLSM analysis were AOB_Beta,

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AOB_eu, NOB_Nba, and DEN_Tb. In case multiple hybridization cycles on the same slide cause

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damage to the particles, three groups of double hybridization (i.e., NSO190–NSO1225 probe for

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AOB_Beta and Nse1472 probe for AOB_eu, NSO190–NSO1225 probe for AOB_Beta and NIT3

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probe for NOB_Nba, and NSO190–NSO1225 probe for AOB_Beta and TBD1419 probe for

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DEN_Tb) were conducted separately. The details of the fluorescent probes and hybridization

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conditions are listed in Table S2 and Section 3 in Supporting Information.

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Fluorescent images of the hybridized composting particles on the slides were taken by a

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confocal microscope (TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany). Lasers at 488

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nm and 543 nm were used to excite the FITC and Cy3 fluorophores, respectively. Image

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segmentation and analysis were performed with Image Pro Plus 6.0 software (Media Cybernetics,

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Rockville, MD, USA). Outputs included the distribution area of AOB_Beta (AAOB_Beta), the

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distribution area of AOB_eu (AAOB_eu), the distribution area of NOB_Nba (ANOB_Nba), and the

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distribution area of DEN_Tb (ADEN_Tb). Two particles were examined for each composting

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sample.

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The particles were also observed by an optical microscope (DM 2500, Leica Microsystems

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GmbH, Wetzlar, Germany). The particle area (Apart) was quantified using Image Pro Plus 6.0

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software. The area ratios of the microbes to the particles were calculated according to

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AAOB_Beta/Apart, AAOB_eu/Apart, ANOB_Nba/Apart, and ADEN_Tb/Apart.

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Statistical analysis and data processing. One-way analysis of variance and least

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significant difference tests were performed using SPSS version 15.0 software (SPSS, Inc.,


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Chicago, IL, USA), with the significance level set at p < 0.05, to determine the significant

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difference in the physicochemical parameters between experiments A and B.

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The correlation between the variables at the pile and particle scales was quantified by

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canonical correspondence analysis (CCA) using Canoco 5.0 software (Microcomputer Power,

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USA) to calculate the following: (1) the correlation between the functional genes of AOB_Beta,

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NOB_Nba, DEN_Tb, nirS + nirK, and nosZ and the concentration of NH4+, NO3–, NO2–, and N2O;

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(2) the correlation between the distribution area of the microbes (i.e., AAOB_Beta/Apart,

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AAOB_eu/Apart, ANOB_Nba/Apart, and ADEN_Tb/Apart) and the concentration of NH4+, NO3–, NO2–, and

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N2O; and (3) the Pearson correlation between N2O emission and other variables (i.e., O2, NH3,

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NH4+, NO2–, NO3–, AOB_Beta abundance, NOB_Nba abundance, DEN_Tb abundance, nirS +

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nirK abundance nosZ abundance, AAOB_Beta/Apart, AAOB_eu/Apart, ANOB_Nba/Apart, and ADEN_Tb/Apart).

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RESULTS AND DISCUSSION

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Evolution of temperature, O2 concentration, NH3 emission, and N2O emission at the

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pile scale. As shown in Figure 1a, both the composting temperature of experiments A and B

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followed the classic four-phase pattern, i.e., the mesophilic (days 0–4), thermophilic (days 4–6),

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cooling (days 6–12), and maturation (days 12–16) phases. The ambient temperature was

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maintained at 23°C. As shown in Figure 1b, the O2 concentration declined dramatically during

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the mesophilic phase because of vigorous metabolism. During the thermophilic phase, the

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composting temperature was higher than 50°C, which could kill most pathogens and thereby

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meet sanitary standards.6 Meanwhile, the O2 concentration was at a low level that provided ideal

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conditions for anaerobic denitrification. The trend of NH3 emission is similar to that of the

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composting temperature. The volatilization of NH3 was enhanced during the mesophilic phase,

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and NH3 emission peaked at around 1150 ppmv during the thermophilic phase. In the later



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phases, NH3 emission gradually decreased. Briefly, NH3 was formed throughout the mesophilic

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and thermophilic phases, which is consistent with the previous research on household waste

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composting.39 N2O emission increased in the mesophilic, thermophilic, and cooling phases,

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respectively. N2O emission started around the middle stage of the composting period when NH3

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emission and the composting temperature began to decline, which agrees with the observations

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of the study of swine manure composting.40 There was N2O emission during the cooling phase

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even though the O2 concentration in the reactor was high, suggesting that N2O emission was not

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only related to the aerobic/anaerobic conditions in the macro-environment but also the

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aerobic/anaerobic conditions in the composting particles.


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(a)

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(b)

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Figure 1. Evolution of temperature and gas concentration during pig manure–wheat straw

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aerobic composting: (a) composting temperature and ambient temperature; (b) oxygen (O2)

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concentration, ammonia (NH3) emission, and nitrous oxide (N2O) emission.

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Note: the values of composting temperature, ambient temperature, and O2 are the means of two



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replicates; the error bars of NH3 and N2O are the standard deviations of six replicates.

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Evolution of NH2OH, NH4+, NO2–, and NO3– at the pile scale. The analytical results

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showed that NH2OH was not detected in any of the composting samples. Wunderlin et al.14

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added NH2OH to sludge reactors and examined the resulting variations of nitrogen-containing

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ions. Their results showed that most of the NH2OH was converted into NO3–, which might be the

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reason for the lack of detection of NH2OH in this study. The evolution of NH4+, NO2–, and NO3–

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during pig manure–wheat straw aerobic composting is illustrated in Figure 2. The increase of the

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composting temperature during the mesophilic phase (days 0–4) accelerated the transfer from

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NH4+ to NH3 and the volatilization of NH3. As a result, the NH4+ concentration decreased

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significantly, which agrees with the research on pig slurry–wheat straw composting.41 The

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correlation between the composting temperature and NH3 emission might occur because the

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equilibrium between NH4+ and NH3 was proportional to the Henry constant that is depending on

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temperature.41 The decline of NO2– during the thermophilic phase (days 4–6) and the late phase

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(days 8–16) implied that NO2– oxidation was promoted, because an increase of NO3– was

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observed between days 4 and 16. The variation of NO3– observed in this study is consistent with

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the study of cattle and sheep manure composting, where an increase in the NO3– concentration

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occurred during the maturation phase, when the composting temperature was close to the

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ambient temperature.42


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Figure 2. Evolution of the concentration of ammonium (NH4+), nitrite (NO2–), and nitrate (NO3–)

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during pig manure–wheat straw aerobic composting.

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Note: the error bars are the standard deviations of six replicates.

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Evolution of bacterial abundance (AOB_Beta, NOB_Nba, DEN_Tb, nirS + nirK, and

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nosZ) at the pile scale and related CCA analysis. The qPCR analysis showed that there was no

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AOA in the composting samples, which is accordant with the study of cattle manure composting;

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the reason might be a high level of O2 concentration or moisture content that would harm the

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metabolism of AOA.8,43,44 Moreover, NOB_Nsp, DEN_AT, and DEN_P were not detected.

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Therefore, NOB_Nba and DEN_Tb were the major NOB and DEN, respectively, during pig

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manure–wheat straw composting. The evolution of AOB_Beta, NOB_Nba, DEN_Tb, nirS + nirK,

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and nosZ and the related CCA analysis is depicted in Figure 3. The concurrence of AOB_Beta,

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NOB_Nba, and DEN_Tb suggested that both the aerobic nitrification of AOB and NOB and the

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anaerobic denitrification of DEN existed in the composting piles. The magnitude of AOB_Beta

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approximates the results found in cattle and pig manure composting, where the AOB_Beta

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abundance ranged between 105–106 copies g–1.43 The NOB_Nba was three orders of magnitude



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lower than AOB_Beta. The bacterial abundance of NOB_Nba dropped between days 4 and 8 and

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soon rebounded, probably because the high temperature led to the inactivation of NOB_Nba

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followed by recovery of the bacteria with decreasing temperature. The trends of nirS, nirK, and

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nosZ agree with the results of cattle manure composting, where nirS generally increased and

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nirK and nosZ decreased first and later increased.5 As shown in Figure 3b, the first two CCA

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axes explained 48.9% of the cumulative variance of the correlation between the bacterial

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abundance (AOB_Beta, NOB_Nba, DEN_Tb, nirS + nirK, and nosZ) and the variables (NH4+,

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NO3–, NO2–, and N2O) with p = 0.002, which demonstrated that these variables could

272

significantly explain the variation of bacterial abundance.45 The distance between AOB_Beta and

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NOB_Nba is shorter than that between AOB_Beta and others, indicating a smaller difference

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between AOB_Beta and NOB_Nba abundance. This might occur because both AOB_Beta and

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NOB_Nba are nitrifiers. The abundance of AOB_Beta was related to NH4+ levels because

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AOB_Beta are the important bacteria in NH4+ oxidation. The high correlation between NOB_Nba

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and NO2– was reasonable because NOB_Nba consumed NO2– in the process of nitrification.

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Recall that nirS + nirK is produced by AOB_eu and/or DEN while nosZ is associated with DEN,

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so the fact that both nirS + nirK and nosZ are closely related to N2O emission (Figure 3b)

280

suggested that the denitrification accomplished by AOB_eu and/or DEN was the key pathway of

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N2O emission. As shown in Figure 3b, the distribution of composting samples varied according

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to different composting time. The composting samples of the early phase (days 0–6) featured

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higher NH4+ concentration and AOB_Beta abundance, which demonstrated strong nitrification

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took place at that time. The samples of the late phase (days 12–16) showed higher NO3–

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concentration, N2O emission, and abundance of nirS + nirK and nosZ suggested that both

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nitrification and denitrification were robust during the late phase of composting, and AOB_eu


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and/or DEN were the dominant bacteria in denitrification.

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(a) qPCR analysis

290 291

(b) CCA analysis

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Figure 3. Evolution of bacterial abundance during pig manure–wheat straw aerobic composting:



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(a) real-time quantitative polymerase chain reaction (qPCR) analysis; (b) related canonical

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correspondence analysis (CCA).

295

Note:

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denitrificans; nirS + nirK, nitrite reductase; nosZ, nitrous oxide reductase; NH4+, ammonium;

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NO2–, nitrite; NO3–, nitrate; N2O, nitrous oxide; the error bars in Figure 3a are the standard

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deviations of six replicates; the sample name in Figure 3b is “composting days-experiment A or

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B-the serial number of the replicates”.

AOB_Beta,

Betaproteobacteria;

NOB_Nba, Nitrobacter;

DEN_Tb,

Thiobacillus

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Dynamic distribution of AOB_Beta, AOB_eu, NOB_Nba, and DEN_Tb in composting

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particles. In the FISH–CLSM images (Figure 4), the dynamic distribution of AOB_Beta,

302

AOB_eu, NOB_Nba, and DEN_Tb in composting particles was qualitatively evaluated. As shown

303

in Figure 4a, AOB_Beta was sporadically distributed on the outer area of the composting

304

particles. Earlier research23 showed the possibility that the outer area of composting particles was

305

aerobic, so it was possible that AOB_Beta therefore conducted aerobic reactions, such as

306

ammonia oxidation. The location of AOB_Beta is similar to the distribution of AOB_Beta in

307

biomass granules,46 but the area of AOB_Beta in composting particles is larger than that in

308

biomass granules. The reason might be either that the microbial communities differentiated

309

between different materials, or that the previous research used a NSO190 probe to stain

310

AOB_Beta, while the present study used a combined NSO190–NSO1225 probe that was more

311

comprehensive. Furthermore, the distribution of AOB_Beta was discontinuous (Figure 4a),

312

which is accordant with the previous CLSM analysis of biofilm.47 However, AOB_eu, which also

313

belongs to the AOB category, was located in the interior of the composting particles (Figure 4a).

314

The different location between AOB_Beta and AOB_eu might be attributed to the spatial

315

difference of NH4+ and NO2– in the composting particles and different functions of the bacteria.


Page 19 of 40


316

Matsumoto et al.48 and He et al.49 determined the profiles of NH4+ and NO2– in sludge and

317

biomass granules using microelectrodes. They found that the NH4+ concentration decreased from

318

the granular surface inwards, while the NO2– concentration showed a slight increase. From this it

319

might be inferred that the NH4+ concentration was higher in the outer area of the composting

320

particles, which facilitated the growth of AOB_Beta in the outer area. In contrast, the NO2–

321

concentration was higher in the interior of the particles, which provided hospitable conditions for

322

AOB_eu. However, this conjecture should be further investigated using microelectrodes or other

323

techniques. As shown in Figure 4b, NOB_Nba were internally attached to AOB_Beta, which is

324

consistent with the biofilm structure seen during wastewater treatment.50 NOB_Nba were

325

adjacent to AOB_Beta, probably because NOB_Nba needed to take in the NO2– generated from

326

AOB_Beta.46,51 Moreover, as NOB_Nba and AOB_Beta competed for the available O2,

327

NOB_Nba lost out and was forced to attach to the AOB_Beta internally.52 As shown in Figure 4c,

328

DEN_Tb was concentrated in the interior of the composting particles, which suggested the

329

denitrification due to DEN was an anaerobic reaction. The coexistence of AOB_Beta and

330

DEN_Tb in the composting particles illustrates a link between the mechanisms of aerobic

331

nitrification and anaerobic denitrification during pig manure–wheat straw aerobic composting.



332 333

(a) AOB_Beta (red) and AOB_eu (green)


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334 335

(b) AOB_Beta (red) and NOB_Nba (green)



336 337

(c) AOB_Beta (red) and DEN_Tb (green)


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338

Figure 4. Dynamic distribution of nitrifiers and denitrifiers in composting particles: (a) Betaproteobacteria (AOB_Beta) and

339

Nitrosomonas europea/Nitrosomonas eutropha (AOB_eu); (b) AOB_Beta and Nitrobacter (NOB_Nba); (c) AOB_Beta (red) and

340

Thiobacillus denitrificans (DEN_Tb).

341

Note: the scale of the images is given on the first image.



342

Evolution of the distribution area of AOB_Beta, AOB_eu, NOB_Nba, and DEN_Tb in

343

composting particles and related CCA analysis. As mentioned above in Materials and

344

Methods, we represent the distribution of the various types of bacteria by calculating the ratio of

345

the area occupied by the bacteria in question to the area of the surface of the particle on which

346

the bacteria has grown. As shown in Figure 5a, AAOB_Beta/Apart is the highest, which agrees with

347

the fact that AOB_Beta had the highest magnitude in the qPCR results above (Figure 3a). The

348

variation of AAOB_eu/Apart in Figure 5a is very similar to the variation of N2O emission shown in

349

Figure 1b, which suggested that AOB_eu was associated with N2O emission. Also, the evolution

350

of ANOB_Nba/Apart shown here agrees with that of NOB_Nba shown in Figure 3a. As shown in

351

Figure 5b, the first two CCA axes explained 50.3% of the cumulative variance of the correlation

352

between the bacterial distribution area (AAOB_Beta/Apart, ANOB_Nba/Apart, AAOB_eu/Apart, and

353

ADEN_Tb/Apart) and the variables (NH4+, NO3–, NO2–, and N2O) with p = 0.002. The distance

354

between AAOB_Beta/Apart and AAOB_eu/Apart was significant although both AOB_Beta and AOB_eu

355

belong to AOB, which quantified the difference in their growth habits and reaction mechanisms.

356

The AAOB_Beta/Apart distribution closely followed that of NH4+ variation while ANOB_Nba/Apart

357

correlated well with variations of NO2– and NO3–, which is consistent to the CCA results of the

358

qPCR analysis (Figure 3b). In addition, the distribution of AAOB_eu/Apart correlated strongly with

359

N2O variation, again suggesting that AOB_eu was the most important pathway of N2O emission.


Page 24 of 40

Page 25 of 40


360 361

(a) FISH–CLSM analysis

362 363

(b) CCA analysis



364

Figure 5. Evolution of the distribution area of Betaproteobacteria (AOB_Beta), Nitrosomonas

365

europea/Nitrosomonas eutropha (AOB_eu), Nitrobacter (NOB_Nba), and Thiobacillus

366

denitrificans (DEN_Tb) in composting particles: (a) fluorescent in situ hybridization–confocal

367

laser scanning microscopy (FISH–CLSM) analysis; (b) related canonical correspondence

368

analysis (CCA).

369

Note: AAOB_Beta, AAOB_eu, ANOB_Nba, and ADEN_Tb represent the distribution area of AOB_Beta,

370

AOB_eu, NOB_Nba, and DEN_Tb, respectively; Apart, the particle area; NH4+, ammonium; NO2–,

371

nitrite; NO3–, nitrate; N2O, nitrous oxide; the error bars in Figure 5a are the standard deviations

372

of four replicates.

373

Regarding the contribution of nitrification, nitrifier denitrification, and anaerobic

374

denitrification to N2O production, the results of multiple linear regression showed N2O = –

375

7.005AAOB_Beta + 544.752AAOB_eu + 159.452ADEN_Tb with a coefficient of determination (R2) of

376

0.87, where the p-values of AAOB_Beta, AAOB_eu, and ADEN_Tb were 0.771, 0.001, and 0.188. This

377

statistics implied that the correlation between nitrifier denitrification and N2O emission was

378

extremely significant (p < 0.01). Although AOB_Beta had the highest abundance (Figure 3a) and

379

distribution area (Figure 5a), the correlation between AAOB_Beta and N2O emission was

380

insignificant. It may be that, as in the case of Wunderlin et al.,14 most of the NH2OH was

381

converted into NO3– but the contribution of nitrification to N2O emission could be relatively low.

382

As shown in Table S3, the Pearson correlation analysis between N2O emission and individual

383

variables (O2, NH3, NH4+, NO2–, NO3–, AOB_Beta abundance, NOB_Nba abundance, DEN_Tb

384

abundance, nirS + nirK abundance nosZ abundance, AAOB_Beta/Apart, AAOB_eu/Apart, ANOB_Nba/Apart,

385

and ADEN_Tb/Apart) also showed that there was a strong positive linear correlation between

386

AAOB_eu/Apart and N2O emission (N2O emission = 1161.7AAOB_eu/Apart, R2 = 0.84). Given the high


Page 26 of 40

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387

correlation between AAOB_eu/Apart and N2O emission observed by multiple linear regression and

388

Pearson correlation analysis, the AOB_eu-driven denitrification, i.e., nitrifier denitrification,

389

appears to be the main pathway of N2O emission. Therefore, during the entire aerobic

390

composting process, one variety of AOB participated in aerobic nitrification while another

391

variety performed nitrifier denitrification. Besides, as shown in Table S3, a positive correlation

392

was found between N2O emission and nosZ, although the correlation was expected to be negative

393

because of anaerobic denitrification. Some previous studies of wetland soils and agricultural

394

soils also found this discrepancy.53,54 The reason for this apparent anomaly is that nitrification by

395

AOB_Beta and nitrifier denitrification by AOB_eu contributed more to N2O emission than did

396

anaerobic denitrification, and since the nosZ gene has not yet been found to be in AOB_Beta and

397

AOB_eu;55-57 the N2O generated during nitrification and nitrifier denitrification must have

398

stimulated growth of DEN_Tb, thereby increasing the concentration of nosZ and generating a

399

positive relationship between N2O emission and nosZ.

400

This study presented the multiple linear regression of N2O emission and demonstrated the

401

important role of AOB_eu to N2O emission. More advanced technology, such as isotope labeling

402

and microelectrodes,14,58,59 could be employed in future studies to measure the contribution of

403

nitrification, nitrifier denitrification, and anaerobic denitrification to N2O emission and to better

404

identify the source of N2O emission during composting.

405

N2O emission mechanisms of pig manure–wheat straw aerobic composting based on

406

multivariate characterization at multi-scale. Based on the multivariate characterization results

407

at both the pile scale and the particle scale (Figures 1b, 2, 3a, and 5a), the nitrification and N2O

408

emission mechanisms of pig manure–wheat straw aerobic composting are summarized in Table 2.

409

Dividing the observation period into the usual three phases (mesophilic, thermophilic, and



410

cooling), we observed growth of N2O emission in all three periods. Comparing the N2O growth

411

with the variation in the concentrations and abundance of ions and microbes across these three

412

periods enables us to draw some conclusions about the nitrification and N2O emission

413

mechanisms taking place during the three phases. The evolution of NO3– and AOB_Beta could be

414

used to reveal the status of nitrification during composting, because NO3– is the final product of

415

nitrification and AOB_Beta dominate the first step of nitrification (i.e., ammonia oxidation).

416

During the mesophilic phase, the concentration of NO3– was almost unchanged while AOB_Beta

417

kept increasing, suggesting that nitrification was not yet completed. During the thermophilic and

418

cooling phases, NO3– increased while AOB_Beta stayed constant at a high level, demonstrating a

419

period of active nitrification. According to previous studies,60,61 the increase in nirS + nirK could

420

be used to identify denitrification due to AOB_eu and/or DEN, because nirS + nirK is the nitrite

421

reductase derived from AOB_eu and/or DEN; since we observed increases in these enzymes in

422

all three phases, we could conclude that denitrification by one or both of these types of bacteria

423

was occurring. In contrast, an increase in nosZ seen in the third phase suggested denitrification

424

due to DEN, because nosZ is the nitrous oxide reductase generated by DEN. The evolution of

425

AAOB_eu/Apart indicated the status of nitrifier denitrification. As shown in Table 2, during the

426

mesophilic and thermophilic phases (days 0–6), the increasing nirS + nirK and AAOB_eu/Apart and

427

the constant nosZ implied that the denitrification of AOB_eu was more active than that of DEN

428

and, therefore, the N2O emission resulted mainly from the denitrification of AOB_eu, i.e.,

429

nitrifier denitrification. During the cooling phase (days 8–12), the rise in nosZ and the reduction

430

in AAOB_eu/Apart suggested a greater degree of anaerobic denitrification due to DEN, and so, over

431

the entire composting cycle, N2O emission was derived from a collaboration of AOB_eu and

432

DEN.


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433

Table 2. Nitrification and nitrous oxide (N2O) emission mechanisms of pig manure–wheat straw

434

aerobic composting based on multivariate characterization at multi-scale. Time

Phase

N2O

NO3–

AOB_Beta

nirS + nirK nosZ

AAOB_eu/Apart

Days 0–4

Mesophilic

↑

-

↑

↑

-

↑

Days 4–6

Thermophilic

↑

↑

-

↑

-

↑

↑

↑

-

↑

↑

↓

Days 8–12 Cooling 435

Note: NO3–, nitrate; AOB_Beta, Betaproteobacteria; nirS + nirK, nitrite reductase; nosZ, nitrous

436

oxide reductase; AAOB_eu, the distribution area of Nitrosomonas europea/Nitrosomonas eutropha;

437

Apart, the particle area.

438

To summarize, during pig manure–wheat straw aerobic composting, AOB_Beta

439

predominated on the outer area of the composting particles, which was exposed to O2, during the

440

first phase of the composting period, which suggested that AOB_Beta conducted ammonia

441

oxidation and generated NO2– aerobically during this phase. In the next phase, NOB_Nba grew

442

and attached internally to AOB_Beta, utilizing NO2– to produce NO3–. In the interior of the

443

composting particles, the anaerobic conditions favored AOB_eu and DEN_Tb to perform

444

denitrification and result in N2O emission. During composting overall, nitrification was

445

consistently carried out; during the mesophilic and thermophilic phases, N2O emission was

446

derived from nitrifier denitrification due to AOB_eu; during the cooling phase, N2O emission

447

was from the denitrification due to both AOB_eu and DEN.

448

ENVIRONMENTAL RELEVANCE

449

According to the results of our multivariate characterization, more attention should be

450

focused on N2O emission during the late phase of composting, which originated from the

451

denitrification of both nitrifiers and denitrifiers. Perhaps more AOB_Beta and NOB_Nba could 29 Environment ACS Paragon Plus


452

be added into composting mixtures to maximize the efficiency of nitrification and to retard

453

denitrification. Improving O2 permeation through the composting particles may also shrink the

454

denitrification area and therefore reduce N2O emission. The statistical significance of the multi-

455

scale quantitative analysis of the variables measured here raises hope that further development of

456

multi-scale models of N2O emission of pig manure–wheat straw aerobic composting may be of

457

some value. For example, the quantitative results for AAOB_Beta/Apart, AAOB_eu/Apart, ANOB_Nba/Apart,

458

and ADEN_Tb/Apart could help to build particle-scale N2O emission models that might be

459

appropriate to laboratory-scale composting systems. And the correlation observed between the

460

particle-scale and pile-scale variables might be useful for scaling the models up and

461

approximating the reality of factory-scale composting. These findings improve our

462

understanding of the mechanisms of N2O emission during manure-based aerobic composting and

463

may provide insights to facilitate decision making regarding responsible strategies for mitigating

464

climate change.

465

The applicability of the multivariate and multi-scale approaches to other waste composting

466

practices, e.g., composting of cattle and cow manures, that are known to lead to production of

467

significant N2O emissions, should be verified and modified in further studies. Furthermore, these

468

systematic methods could be useful for investigating the mechanisms of other greenhouse gas

469

emissions and odors, such as methane (CH4) and hydrogen sulfide (H2S), although the ions and

470

microbes involved may differ and may have to be specifically characterized. The potential

471

competition between and among these microbes should also be addressed. For instance, acetate,

472

sulfate, sulfite, and sulfide are the key ions involved in CH4 and H2S emissions; there is synergy

473

and competition that needs to be investigated in future work on nitrifiers, denitrifiers,

474

methanogens, methanotrophs, sulfate-reducing bacteria, and sulfur-oxidizing bacteria.7,62-64


Page 30 of 40

Page 31 of 40


475 476

CORRESPONDING AUTHOR

477

Lujia Han

478

China Agricultural University (East Campus), Box 191, Beijing 100083, China

479

Fax: 86-10-6273-6778

480

Tel: 86-10-6273-6313

481

E-mail: [email protected]

482 483

ACKNOWLEDGMENTS

484

This work was financially supported by the China Agriculture Research System (CARS-36),

485

the Program for Changjiang Scholars and Innovative Research Team in University (IRT1293),

486

and the National Natural Science Foundation of China (31771684). We thank Christopher

487

Monroe for editing support.

488 489

SUPPORTING INFORMATION AVAILABLE

490

Section S1. Details of real-time quantitative polymerase chain reaction (qPCR).

491

Table S1. Primers used in qPCR analysis.

492

Section S2. Preparation of particle samples for fluorescent in situ hybridization–confocal laser

493

scanning microscopy (FISH–CLSM) analysis.

494

Figure S1. Micro-CT images and thickness of the composting particles.

495

Table S2. Fluorescent probes and hybridization conditions.

496

Section S3. Hybridization details.

497

Table S3. Pearson correlation analysis between nitrous oxide (N2O) emission and other variables.



498 499

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