Nitrogen Removal and N2O Accumulation during Hydrogenotrophic

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Nitrogen removal and NO accumulation during hydrogenotrophic denitrification: influence of environmental factors and microbial community characteristics Peng Li, Yajiao Wang, Jiane Zuo, Rui Wang, Jian Zhao, and Youjie Du Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00071 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 2, 2016

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Environmental Science & Technology

1

Nitrogen removal and N2O accumulation during hydrogenotrophic

2

denitrification: influence of environmental factors and microbial

3

community characteristics

4

Peng Li1, Yajiao Wang1, Jiane Zuo*, Rui Wang, Jian Zhao, Youjie Du

5

State Key Joint Laboratory of Environmental Simulation and Pollution Control,

6

School of Environment, Tsinghua University, Beijing 100084, China

7

*

8

(86)-10-62772455

9

1

Corresponding author, Email address: [email protected], Phone number:

These authors contributed equally to this work.

10

Abstract

11

Hydrogenotrophic denitrification is regarded as an efficient alternative technology of

12

removing nitrogen from nitrate-polluted water that has insufficient organics material.

13

However, the biochemical process underlying this method has not been completely

14

characterized, particularly with regard to the generation and reduction of nitrous oxide

15

(N2O). In this study, the effects of key environmental factors on hydrogenotrophic

16

denitrification and N2O accumulation were investigated in a series of batch tests. The

17

results show that nitrogen removal was efficient with a specific denitrification rate of

18

0.66 kg N/(kg MLSSd), and almost no N2O accumulation was observed when the

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dissolved hydrogen (DH) concentration was approximately 0.40 mg/L, the

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temperature was 30 °C and the pH was 7.0. The reduction of nitrate was significantly

21

affected by the pH, temperature, inorganic carbon (IC) content and DH concentration.

ACS Paragon Plus Environment


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A considerable accumulation of N2O was only observed when the pH decreased to 6.0

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and the temperature decreased to 15 °C, where little N2O accumulated under various

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IC and DH concentrations. To determine the microbial community structure, the

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hydrogenotrophic denitrifying enrichment culture was analyzed by Illumina

26

high-throughput sequencing, and the dominant species were found to belong to the

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genera Paracoccus (26.1%), Azoarcus (24.8%), Acetoanaerobium (11.4%), Labrenzia

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(7.4%), and Dysgonomonas (6.0%).

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Key words: hydrogenotrophic denitrification; nitrous oxide accumulation; microbial

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community; high-throughput sequencing; kinetics

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TOC art 1. Introduction

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Nitrate is one of the most critical pollutants in water and wastewater, and nitrate

35

pollution of drinking water might increase the risk of non-Hodgkin's lymphoma and


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bladder and ovarian cancers in humans.1 Nitrate loading to surface water is commonly

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considered an important contributor to water quality deterioration and eutrophication.2

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Biological denitrification technologies have been widely applied for nitrogen removal

39

because they provide efficient performance and low cost.3,4,5,6 Hydrogenotrophic

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denitrification is an innovative, effective and clean technology that has attracted

41

considerable attention recently, and its main advantage over heterotrophic

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denitrification is that an additional dosage of

43

required.7,8

44

denitrification for the treatment of nitrate-polluted water with insufficient bioavailable

45

organics, such as groundwater, drinking water and secondary effluent of

46

wastewater.9,10

This

property

an organic carbon source is not

increases the effectiveness

of

hydrogenotrophic

47

A variety of high-efficiency bioreactors of hydrogenotrophic denitrification have

48

been developed, such as fixed- and fluidized-bed reactors9,11, membrane biofilm

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reactors and biofilm electrode reactors12,13. Because of the low solubility of hydrogen

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and the risk of explosion with escaped hydrogen gas, improving the efficiency and

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security of hydrogen delivery remains a major challenge. Therefore, many innovative

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hydrogen delivery systems have been developed along with membrane biofilm

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reactors using gas-permeable membranes including hollow-fiber or silicon tubes.10

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In addition to the advancements in reactor configuration, a better understanding

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of the inherent biochemical process underlying hydrogenotrophic denitrification may

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also help promote the technology. Several kinetic models have been proposed to



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reveal the regularity of the process of hydrogenotrophic denitrification.14,15,16 In these

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studies, hydrogenotrophic denitrification was regarded as a two-step consecutive

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reduction of nitrate to nitrite and then to nitrogen gas. However, the complete process

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of hydrogenotrophic denitrification is actually a four-step process in which hydrogen

61

supplies electrons for a stepwise reduction of nitrate to nitrite, nitric oxide (NO),

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nitrous oxide (N2O) and nitrogen gas through a series of reductases. When the

63

production rate is higher than the consumption rate, N2O accumulates in the liquid

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phase, which causes the emission of N2O into the atmosphere. N2O is known as a

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main greenhouse gas, and biological denitrification during wastewater treatment is

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considered as one of the anthropogenic sources of N2O emission.

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evaluation of the characteristics of N2O accumulation, which depends on the

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microbial community and electron donor and environmental factors, is significant to

69

mitigating N2O emissions from wastewater treatment. Although the characteristics of

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N2O accumulation during heterotrophic denitrification have been comprehensively

71

investigated,18,19 related research on hydrogenotrophic denitrification is not available.

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This research deficit reduces our ability to understand the process of

73

hydrogenotrophic denitrification.

18

Therefore, an

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To analyze the mechanisms underlying the hydrogenotrophic denitrification

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process, further studies are required, including investigations on N2O emissions, as

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well as on the effects of the environmental factors. Previous studies have indicated

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that the effectiveness of hydrogenotrophic denitrification is impacted by several


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crucial environmental factors, including the pH,14,20,21,22 temperature,10,11,20 inorganic

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carbon to nitrogen ratios (IC/N),13,22 dissolved hydrogen concentration (DH)13 and the

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nitrate concentration11,23Among these factors, pH has attracted the most interest, and

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the effects of pH in the range of 7.0 to 8.5 on nitrate removal have been

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studied.15,20,21,22 Although consensus on the optimal value has not been reached, the

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high dependency of pH on nitrate reduction and nitrite accumulation has been

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illustrated, which implies the significance of pH on N2O reduction and accumulation.

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In most cases, researchers investigating the nitrogen removal efficiency of

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hydrogenotrophic denitrification bioreactors have focused on the effects of the

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operational parameters, such as the hydrogen supply interval and the inorganic carbon

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dosage methods.13,21 Although the potential role of influential factors was indirectly

89

suggested by these studies, the inherent effects of these factors on the biochemical

90

process were not revealed. In addition, because the microbial cultures utilized in

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various studies were different and the effects of multi factors were rarely involved in

92

one study, it is difficult to perform comparisons among factors across experiments.

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Therefore, a comprehensive study must be performed to unveil the effects of multi

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environmental factors on the biochemical process of hydrogenotrophic denitrification.

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The objectives of this paper are as follows: 1) to determine the rate of N2O

96

reduction and accumulation in hydrogenotrophic denitrification; 2) to systemically

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investigate how the key environmental factors (i.e., pH, temperature, inorganic carbon

98

concentration and DH concentration) affect nitrate reduction, nitrite accumulation and



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N2O emission; and 3) to determine the microbial community of hydrogenotrophic

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denitrifiers by Illumina high-throughput sequencing. This study will help provide a

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better understanding of the hydrogenotrophic denitrification process, and the results

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will have important implications for the implementation and operation of

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hydrogenotrophic denitrification technology.

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2. Methods and Materials

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2.1 Bacteria acclimation and cultivation

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The initial inoculum of the hydrogenotrophic denitrifying culture was the

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activated sludge from the aerobic tank (Xiaojiahe Municipal Wastewater Treatment

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Plant, Beijing, China). After 60 days of constant feeding with H2 and synthetic

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nutrition, the denitrification rate remained constant. Therefore, it was confirmed to

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achieve a steady state for enrichment cultures. The enrichment culture was cultivated

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for 54 months continuously and then served as the stock culture. The acclimation and

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cultivation were conducted in batch mode at pH 7.0±0.5 at 30±1°C, with a cycle time

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of 12 h using three parallel parent reactors with a working volume of 2.0 L. Hydrogen

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gas was produced in situ through water electrolysis (HG-1805, Beijing, China).

115

Synthetic water was used as feed, and it contained (per liter) 1.62 g KNO3, 0.5 g

116

NaHCO3, 0.55 g NaH2PO4·2H2O, 2.11 g K2HPO4·3H2O and several other necessary

117

nutrients, including 7.3 mg CaCl2·2H2O, 5.0 mg FeSO4·7H2O, 2.5 mg MnCl2·4H2O,

118

0.5 mg CoCl2·6H2O, 0.5 mg (NH4)6Mo7O24·4H2O, 0.22 mgZnSO4·7H2O and 0.2 mg

119

CuSO4·5H2O.


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2.2. Batch experiments

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2.2.1 Reactors

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Each batch test in this study was repeated three times in parallel. The batch

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reactor was sealable and the working volume of the reactor was 600 ml with 450 ml

124

of headspace. The hydrogen was transferred from the headspace to the liquid bulk

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through a gas-liquid interface. At the beginning of a test, 140 ml of the synthetic feed

126

water was added to each batch reactor, and then 10 ml of the centrifugal stock culture

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taken from the parent reactors was inoculated. The hydrogen gas produced by

128

electrolysis was utilized to ensure that the volume percentage of the hydrogen in the

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headspace was 98~100% by alternating headspace evacuation of the reactor vacuum

130

with hydrogen gas input.

131

KNO3, KNO2 and N2O were used as substrates of denitrification, and NaHCO3

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was used as the inorganic carbon source at the given concentration. The synthetic

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contaminated water (per liter) also contained 0.55 g NaH2PO4·2H2O and 2.11 g

134

K2HPO4·3H2O as buffers.26

135

The pH was controlled during the tests by adding 0.2 M HCL and 0.2 M NaOH

136

solutions to the set point ± 0.2. The temperature of the reaction system was

137

maintained at the set point ± 0.5 °C by a temperature adjustable shaking incubator

138

with a rotation speed of 130 r/min.

139 140

All of the batch tests conditions are listed in Table S1. 2.2.2 Batch test I: reduction performance of nitrate, nitrite and N2O



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Nitrate, nitrite and N2O were utilized as the sole electron acceptors with an

142

adequate electron donor (hydrogen). The initial NO3--N and NO2--N concentrations

143

were controlled at approximately 40 mg/L using KNO3 and KNO2. To control the

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initial N2O-N concentration, pure N2O gas (99.99%) was injected into the reactor to

145

replace the volume of hydrogen gas to provide the initial nitrogen concentration of 60

146

mg/L after normalizing to the liquid volume according to Henry’s Law. Batch test I

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was conducted under a pH of 7.0, a temperature of 30°C, an initial mass ratio of IC/N

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of 1.8 and a volume percentage of hydrogen gas at 100% in the headspace.

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The N2O-N concentration in this study indicates the sum of the N2O-N

150

concentration in both the gaseous and liquid phases after normalizing to the liquid

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volume. The N2O-N or NO2--N accumulation during denitrification was estimated by

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the accumulation percentage, which is described as the ratio of the maximal

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accumulation concentration of nitrogen contained in N2O or NO2- to the initial

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nitrogen concentration.

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2.2.3 Batch test II: effects of pH, temperature, inorganic carbon concentration

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and DH concentration on hydrogenotrophic denitrification

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Batch test II was conducted to study the influence of the pH, temperature,

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inorganic carbon content and DH concentration on hydrogenotrophic denitrification

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with an initial NO3--N of approximately 40 mg/L.

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In the pH tests, the pH was maintained at 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 under

161

temperature of 30 °C, IC/N mass ratio of 1.8 and DH concentration of 0.40 mg/L. An


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overdose of inorganic carbon was used to ensure of a sufficient carbon supply. In the

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temperature tests, the temperature was maintained at 15, 20, 25, 30, 35, 40, 45 and

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50 °C under pH of 7.0, IC/N mass ratio of 1.8 and DH concentration of 0.40 mg/L. In

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the inorganic carbon concentration tests, the initial mass ratio of IC/N was adjusted to

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0, 0.18 and 1.8 under pH of 7.0, temperature of 30 °C, and DH concentration of 0.40

167

mg/L. To study the effect of the hydrogen concentration, pure argon gas was used to

168

replace the volume of hydrogen gas to adjust the initial gaseous volume percentage of

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hydrogen in the headspace to 10%, 50% and 100%, under pH of 7.0, temperature of

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30 °C and IC/N mass ratio of 1.8.

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approximately 0.02, 0.17 and 0.40 mg/L, respectively.

172

2.3 Sampling and analytical methods

The DH concentrations were determined to reach

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The gaseous samples were collected in gasbags for further analysis of the

174

gaseous N2O and H2. The liquor samples were used for the analyses of nitrite, nitrite,

175

dissolved N2O and DH after filtering through a disposable Millipore filter (0.45 µm

176

pore size). MLSS samples were collected at the end of each experiment and

177

determined by weighing the dry cell. The intervals of the sampling were between 10

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min to 3 h according to the rate of reaction.

179

The gaseous hydrogen was analyzed by gas chromatography (GC) (Agilent 7890,

180

USA) with a Carbonxen-1000 packed column (1.5 m×2.1 mm×60/80 mesh, Supelco)

181

and a thermal conductivity detector (TCD). Gaseous N2O was measured by GC

182

(Agilent6890N, USA) using a

Porapak Q packed column (1.8 m×2.1 mm×80/100



183

mesh, Supelco) and an electron capture detector (ECD).

184

The DH concentration was measured by the headspace method.26Liquor samples

185

were taken with headspace vials that were then immediately sealed and vigorous

186

shaken and resting for more than 2 hours. Thus, the hydrogen in the gaseous and

187

liquid phase almost achieved its equilibrium state. The hydrogen concentration in the

188

headspace of the vial was measured by GC according to the methods described above.

189

The liquor sample concentration would be calculated according to Henry’s Law. A

190

similar method was also utilized to measure the dissolved N2O concentration. The

191

gaseous N2O concentration of the headspace inside the vial was measured by GC. The

192

dissolved N2O concentration was calculated according to Henry’s Law and the Mass

193

Conservation Law. The DH and dissolved N2O were calibrated according to the

194

method of Lu et al.24 The detection limits for the DH and dissolved N2O were below

195

0.001 mg/L and 0.002 mg/L, respectively, and the coefficients of variation were

196

0.0056 and 0.0112, respectively.

197

The nitrate, nitrite and MLSS were determined according to the standard

198

methods of the Chinese NEPA.25 The pH was determined by portable meters (HQ40D,

199

Hach, USA). All of the samples were analyzed in triplicate.

200

2.4. Microbial community analysis

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2.4.1 DNA extraction and PCR amplification

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The genomic DNA of the enriched cultures of the hydrogenotrophic denitrifiers

203

was extracted using the E.Z.N.A.Soil DNA Kit D5625-01 (OMEGA, USA). The


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purity and concentrations of the nucleic acids were accurately measured using a Qubit

205

2.0 fluorometer (Life Tech, USA). The V3-V4 hypervariable regions of the bacterial

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16S

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(5’-CCTACGGGNGGCWGCAG-3’)

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ATCC-3’).The PCR products were pooled and purified with a SanPrep Column DNA

209

Gel Extraction Kit SK8192 (Sangon Biotech, China). The details of the PCR

210

amplification are available in the Supplemental Material.

211

2.4.2 Illumina high-throughput sequencing and data analysis

rRNA

genes

were

amplified and

805R

with

the

primers

341F

(5’-GACTACHVGGGTATCTA

212

The DNA library was prepared according to the MiSeqTM Reagent Kit

213

Preparation Guide (Illumina, San Diego, CA, USA) and run on a MiSeq Illumina

214

platform for sequencing (Sangon Tech, China). The read preprocessing, operational

215

taxonomic unit (OTU) generation and identification, and statistical analyses were

216

performed with Mothur, and the taxonomic classification was assigned using the RDP

217

Classifier. The OTUs were generated with an identity threshold of 97%.

218

3. Results and Discussion

219

3.1 Nitrate, nitrite and N2O reduction by hydrogenotrophic denitrifiers

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The reduction rates of nitrate and its intermediates were evaluated using nitrate,

221

nitrite and N2O as the sole electron acceptor. As shown in Figure 1, all of the

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concentrations of NO3--N, NO2--N and N2O-N/L linearly declined to depletion. The

223

specific reduction rates of NO3--N, NO2--N and N2O-N, were calculated to be 0.66,

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0.76 and 3.96 kg N/(kg MLSSd), respectively. The nitrate reduction rates were higher



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than those of previous studies under similar conditions, which were in the range from

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0.38 to 0.60 kg N/(kg MLSSd).14,15,20 The results indicated that the enriched cultures

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in this study were highly efficient in hydrogenotrophic denitrification. Although the

228

reference of N2O reduction rate from other research is still unavailable at present, it is

229

comparable with that in heterotrophic denitrification. 26

230

The specific reduction rate of NO2--N was determined to be higher than that of

231

NO3--N. Theoretically, it is reasonable to anticipate that nitrite would not be observed

232

during the batch test. However, when using nitrate as the sole electron acceptor, a

233

slight nitrite accumulation was observed with a maximum of 5.0 mgN/L, which then

234

rapidly disappeared. Rezania et al. observed that the nitrite reduction rate declined in

235

the presence of nitrate.14 The reduction of both nitrate and nitrite is known to occur at

236

the end of the respiratory chain. Thus, competition for the hydrogen carrier and

237

electron carrier might occur between nitrate reductase and nitrite reductase. Pan et al.

238

demonstrated the hypothesis of electron competition in heterotrophic denitrification

239

by methanol utilizing denitrifiers.27 The competitive inhibition is a reasonable

240

explanation of nitrite accumulation during nitrate reduction.

241

When using nitrate as sole electron acceptor, N2O was detected at an extremely

242

low concentration of less than 0.018 mg N2O-N/L, which accounted for only 0.044%

243

of the initial NO3--N concentration. This result indicated that N2O accumulation could

244

be controlled at an extremely low level during hydrogenotrophic denitrification.

245

When using nitrite as the sole electron acceptor, N2O accumulation was observed and


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achieved a maximum at the beginning of the reaction, and then gradually decreased to

247

depletion. The maximum concentration of N2O-N accumulated was 0.27 mgN/L,

248

which accounted for 0.75% of the initial NO2--N concentration. Moreover, the N2O

249

accumulation was obviously higher than that when using nitrate as the sole electron

250

acceptor. Hence, it could be confirmed that N2O reduction would be inhibited in the

251

presence of nitrite with relative high concentration. Adverse impact of nitrite on N2O

252

reduction has been widely reported in the studies on heterotrophic denitrification.18

253

3.2 Effect of pH on denitrification and N2O production

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Nitrate reduction and N2O generation were investigated at different pH values,

255

and the results are shown in Figure 2. The effects of pH on hydrogenotrophic

256

denitrification were significant. The specific nitrate reduction rate gradually increased

257

with the increase in the pH value from 6.0 to 8.0, and achieved at maximum of 0.90

258

kg N/(kg MLSSd) at pH 8.0, which gradually decreased when the pH increased from

259

8.0 to 9.0 (Figure 2(b)). The nitrite accumulation achieved a minimum at a pH of 8.0,

260

and gradually increased when the pH decreased from 8.0 to 6.0 or increased from 8.0

261

to 9.0. It achieved 13.6 and 8.4 mg/L at the pH of 6.0 and 9.0, respectively. The nitrite

262

reduction rate also achieved a maximum at pH 8.0, and declined when the pH

263

changed from this value. The results indicate that the pH dependency of nitrate is

264

similar to that of nitrite in the pH range of 6.0 to 9.0. There is an optimal pH value for

265

both of nitrate and nitrite reduction, and the reduction rates decrease when the pH

266

deviates from this value. Nitrite accumulation was likely caused by the lower

267

reduction rate of nitrite relative to that of nitrate. Hence, it could be inferred that



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nitrite reductase is more sensitive to pH variation than nitrate reductase. Similar

269

results were reported by Ghafari et al., which demonstrated that the optimal pH value

270

for nitrate reduction was in the range of 7.5 to 8.0 and the highest specific reduction

271

rate was 0.53 mg N/(g MLSSd).16 However, Rezania et al. obtained different results,

272

demonstrating that the specific nitrate and nitrite reduction rates gradually increased

273

as the pH increased and nitrite accumulation did not occur at pH value from 7.5 to

274

9.5.14The inconsistency in the optimal pH for hydrogenotrophic denitrification might

275

have been related to the different cultures used in the experiments. The cultures

276

enriched under different cultivation conditions would form different microbial

277

community structures, which result in different denitrification characteristics.

278

The report on the N2O accumulation during hydrogenotrophic denitrification is

279

still unavailable at present. It was reported that for heterotrophic denitrification with

280

the initial ratio of organic carbon to nitrogen varying from 3.5 to 10 (mol/mol), the

281

concentration of N2O in liquid phase is in the range from 0.1 to 3.0 mg N/L

282

What’s more, the conversion ratio of N2O-N concentration to initial NO3--N

283

concentration were reported in the range from 0 to 14% when organic carbon was

284

abundant for denitrification.

285

concentration accumulated in liquid phase was observed extremely low, less than

286

0.022 mgN/L at pH in the range from 7.0 to 9.0, corresponding to only 0.056% of the

287

initial NO3--N concentration. Therefore, we can conclude that the N2O accumulation

288

during hydrogenotrophic denitrification was comparable with heterotrophic

30~33

28,29

.

As shown in Figure 2(C), maximal N2O


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denitrification, and the potential of N2O emission in practice would be acceptable.

290

However, when the pH decreased to 6.5, N2O accumulation increased to 0.163 mg/L.

291

Furthermore, N2O immensely accumulated and achieved 5.84 mg N2O-N/L at pH 6.0,

292

corresponding to 15.2% of initial NO3--N concentration. These results indicated that

293

the N2O reductase could keep highly active at neutral and slightly alkaline condition,

294

and in contrast, be inhibited at slightly acidic condition. It is generally considered that

295

the activity of enzyme will decrease when the pH value deviates from the suitable

296

range. Under slightly acidic condition, N2O massively accumulated during the batch

297

test, indicating that N2O reduction suffered a more serious decline than nitrite

298

reduction. A potential explanation was proposed that N2O reductase could be

299

significantly inhibited by free nitrous acids (FNA). Nitrous acid is a weak electrolyte.

300

Consequently, FNA would be formed in the presence of nitrite at acidic condition. In

301

contrary to none report on the characteristics of N2O accumulation during

302

hydrogenotrophic denitrification, it has been observed that, for heterotrophic

303

denitrification, the amount of N2O accumulation would sharply increase when pH was

304

adjusted to lower than 7.0.34 It is reported that N2O reductase is more severely

305

inhibited than nitrite reductase even if the FNA concentration in solution is as low as

306

0.0007 mgHNO2-N/L.26 The proposed mechanisms of FNA inhibition include

307

uncoupling effect and competitive inhibition to the enzyme. FNA could increase the

308

proton permeability through cell membranes, so that proton motive force would

309

collapse and the synthesis of adenosine triphosphate (ATP) would be inhibited.26



310

Moreover, the active sites of N2O reductase is more easily combining with FNA rather

311

than N2O. 26 As shown in Figure 2(b), the maximal concentration of NO2--N reached

312

13.6 mg/L at pH 6.0. Based on theoretical calculation, FNA concentration would

313

achieve 0.032 mgHNO2-N/L. It is reasonable to conclude that FNA inhibition play an

314

importance role in the N2O accumulation at slightly acidic condition.

315

3.3 The effect of temperature on denitrification and nitrous oxide production

316

Temperature significantly affects the performance of heterotrophic denitrification

317

as demonstrated by extensive researches.35 The effect of temperature on

318

hydrogenotrophic denitrification has not yet been comprehensively evaluated. The

319

temperature dependency of hydrogenotrophic denitrification was investigated at

320

different temperatures in the range of 15.0 to 50.0 °C. The profiles of NO3--N, NO2--N

321

and N2O-N are shown in Figure 3. The apparent optimal temperature was 40 °C for

322

nitrate reduction, with a specific nitrate reduction rate of 2.49 kg N/(kg MLSSd). The

323

specific nitrate reduction rate decreased gradually when the temperature moved away

324

from the optimal value. At the temperature as high as 50 °C, the specific nitrate

325

reduction rate reached 1.29 kg N/(kg MLSSd). When the temperature dropped down

326

to 15 °C, the specific nitrate reduction rate decreased to 0.17 kg N/(kg MLSSd),

327

which corresponded to only 6.6% of that at the optimal temperature. The effect of

328

temperature from 15 °C to 30 °C was previously evaluated through continuous

329

operating bioreactor.10, 36 The results show that higher temperature led to a higher

330

denitrification rate in this range, and indicate that the temperature dependence of the


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denitrification rate could be described by an Arrhenius equation.10 A consensus has

332

not been reached on the optimal temperature. Consistent with the results of this study,

333

Kurt et al. found that the maximum denitrification rate could be obtained at 42 °C.20

334

However, Zhou et al. suggested the suitable temperature range was 30 °C to 35 °C.37

335

Nitrite accumulation was observed at all of the test temperatures. The maximum

336

nitrite accumulation concentration was relatively low at less than 6.5 mg/L, at the

337

temperatures from 20 °C to 35 °C. NO2--N accumulated to a great degree when the

338

temperature fell to 15 °C or exceeded 40 °C. The maximum accumulation

339

concentration of NO2--N achieved to 17.0 and 20.4 mg/L occurred at temperatures of

340

15 °C and 40 °C, respectively. If the nitrite reduction rate were lower than the nitrate

341

reduction rate, NO2--N accumulation would occur. The accumulation concentration of

342

NO2--N was observed increasing when the temperature was deviated from the

343

optimum, indicating that comparing with nitrate reduction rate, the nitrite reduction

344

rate was more affected by temperature. Rezania et al. compared the characteristics of

345

hydrogenotrophic denitrification at 12±1 °C and 25±1 °C and found that nitrate

346

reduction showed stronger competition ability for electron donors than nitrite

347

reduction at low temperatures, which caused unexpected nitrite accumulation at

348

12 °C.14

349

During the experiments at temperatures from 20 °C to 50 °C, the N2O

350

concentration was always maintained at a low level. The maximum N2O-N

351

concentrations at the different temperatures in this range were all below 0.06 mg/L.



352

However, when the temperature fell to 15 °C, the maximum N2O accumulation

353

sharply increased to 2.48 mg N2O-N /L, which corresponded to 6.2% of the initial

354

NO3--N. When the temperature is lower than the suitable range, the activity of enzyme

355

that catalyzes biochemical reactions in microbial cells will decline rapidly. The

356

suitable ranges are different depending on the types of enzyme. As shown in Figure

357

3(C), when the temperature dropped to 15°C, an abrupt increase of N2O accumulation

358

was observed during hydrogenotrophic denitrification, indicating that N2O reduction

359

rate was lower than that of nitrite and nitrate. This result suggested that the

360

temperature of 15°C had been beyond the suitable range for N2O reductase. The

361

temperature ranges in which nitrite and nitrate reductases are active seem to be larger

362

than that for N2O reductase. Although references are not available for

363

hydrogenotrophic denitrification, N2O accumulation has been reported to significantly

364

increase at low temperatures during winter because of the low temperature.

365

3.4 Effect of the IC/N ratio on denitrification and N2O production

366

Hydrogenotrophic denitrifiers have been shown to use inorganic carbon to

367

synthesize their cells through an autotrophic pathway.8 To investigate the effect of the

368

inorganic carbon concentration on denitrification performance, the sequencing batch

369

tests were conducted with an IC/N mass ratio of 0, 0.18 and 1.8, which were roughly

370

equivalent to the stoichiometric amounts of 0, 1 and 10 times (Eq. 5), respectively.

371

The pH was adjusted and maintained at 7.0 for each test, so that the influence of pH

372

could be avoided. As shown in Figure 4, the denitrification performance was

373

promoted by an inorganic carbon source substrate. The specific nitrate reduction rates


Page 18 of 40

Page 19 of 40


374

were determined to be 0.38, 0.51 and 0.66 kg N/(kg MLSSd), which corresponded to

375

IC/N mass ratios of 0, 0.18 and 1.8, respectively. Denitrification was not terminated

376

without a dose of the inorganic carbon source substrate, although carbon dioxide

377

could be fixed and utilized for assimilation, which may explain why the dissolution of

378

carbon dioxide in water supported the inorganic carbon requirement.22

379

Nitrite accumulation was observed in all of three tests. The maximum

380

accumulation concentration of NO2--N was 6.8 mgN/L, which occurred under an IC/N

381

mass ratio of 0.18. The nitrous oxide concentrations were all very low (less than 0.02

382

mgN/L, corresponding to 0.05% of the initial NO3--N) under the various IC/N ratios,

383

indicating that the concentration of inorganic carbon source substrate had little

384

influence on the N2O accumulation in hydrogenotrophic denitrification. Hence, the

385

results demonstrate that N2O reduction rate is always higher than nitrite reduction rate

386

under various IC/N ratios in this study.

387

3.5 Effect of the hydrogen concentration on denitrification and N2O production

388

The effect of the DH concentration on the denitrification performance was

389

evaluated by controlling the volume percentage of hydrogen in headspace. The

390

concentrations of DH, NO3--N, NO2--N and N2O-N during the batch tests are shown

391

in Figure 5. In each of the three batch tests, the DH concentration was maintained at a

392

relatively stable range before nitrate was depleted. DH concentrations of

393

approximately 0.02, 0.17 and 0.40 mg/L corresponded to volume percentage of

394

hydrogen in the headspace of 10%, 50% and 100%, the DH concentrations were



395

determined to reach approximately 0.02, 0.17 and 0.40 mg/L, respectively. After

396

nitrate was reduced completely, the DH concentration gradually increased. The nitrate

397

reduction rate decreased with the hydrogen percentage in headspace decreasing from

398

100% to 10% (shown in Table S1). Similar observation was obtained by Karanasios et

399

al.

400

denitrification performance through continuous operation of bioreactors. With gas

401

flow of hydrogen decreasing, the DH concentration and the denitrification efficiency

402

both decreased.

38

who studied the effects of hydrogen gas supply on the hydrogenotrophic

403

In this study, even though the DH concentration was as low as 0.02 mg/L, the

404

nitrate reduction rate remained at 0.26 kg N/(kg MLSS·d). It was still acceptable in

405

practical application, comparing to the denitrification rates obtained in other studies.

406

39,40

407

very low DH concentration. This phenomenon could be explained by kinetic analysis

408

through a double Monod model. According to the approach described by Lu et al., the

409

hydrogen saturation constant was calculated to be 0.03 mg/L (R2 = 0.963). When DH

410

of 0.02 mg/L was close to saturation constant, the nitrate reduction rate was still

411

satisfactory although the corresponding hydrogen percentage in headspace was only

412

10%.

It is evident that hydrogenotrophic denitrification could be effectively operated at

413

Nitrite accumulation was observed in the batch tests with DH concentration of

414

0.17 and 0.40 mg/L. In hydrogenotrophic denitrification, various reductases of

415

nitrogen oxides could obtain electron from hydrogen. Although the characteristics of


Page 20 of 40

Page 21 of 40


416

the electron distribution among different reductases has not been studied for

417

hydrogenotrophic denitrification, the electron competition among reductases of

418

nitrogen oxides in heterotrophic denitrification was observed by Pan et al. 27 They

419

found that when the electron donors were limited, the competition would be

420

intensified. If the reductase was more competitive, the reduction rate of the

421

corresponding nitrogen oxide would be higher. Therefore, it can be inferred that with

422

limited hydrogen supply, the nitrite reductase is more competitive comparing to

423

nitrate reductase.

424

As shown in Figure 5(d), the characteristics of N2O accumulation were similar at

425

the various DH concentrations, and all of the maximum concentrations were less than

426

0.03 mgN2O-N/L. This result indicated that N2O emission would not increase even if

427

the hydrogen were insufficient. Although limited information is available on the effect

428

of electron donor concentration on N2O reduction for the hydrogenotrophic

429

denitrification, the characteristics of N2O accumulation under electron donor

430

limitation for heterotrophic denitrification has been widely investigated.18 According

431

to the previous reports, when concentration of electron donor was lower than the

432

theoretical quantity demanded for heterotrophic denitrification, the N2O would

433

accumulate significantly in solution.

434

accumulated during hydrogenotrophic denitrification, although at most time, the DH

435

concentration was much lower than theoretical quantity demanded for denitrification

436

corresponding to the nitrate concentration in solution. Moreover, the decrease of DH

18

On the contrary, the N2O was not excessively



Page 22 of 40

437

concentration low to 0.02 mg/L would not cause excessive N2O accumulation. This

438

feature offers a unique advantage for hydrogenotrophic denitrification in reducing

439

N2O emission from nitrogen removal treatment.

440

3.6 Microbial community of hydrogenotrophic denitrifying culture

441

We obtained 84930 high quality sequences with the average length of 445 bps for

442

the analysis of the microbial community. The number of OTUs, Shannon-Weiner

443

Index, ACE and Chao1 richness estimators were 2493, 4.38, 2539.3 and 2495.9.

444

Rarefaction curve plotting OTUs numbers was shown in Supplementary Figure S1.

445

The phylogenetic structure of the hydrogenotrophic denitrifier community was

446

characterized at the phylum, class and genus level, respectively. The relative

447

abundances of the main phyla, classes and genera with sequence percentage more

448

than 0.5% are shown in Figure 6. At the phylum level, the culture was dominated by

449

Proteobacteria

450

Euryarchaeota (4.4%). As shown in Figure 6(b), the OTUs belonging to

451

Proteobacteria were mostly

affiliated to Alphaproteobacteria

452

Betaproteobacteria

although

453

Gammaproteobacteria (0.6%).

(64.0%),

Firmicutes

(25.4%),

(17.4%),

they

Bacteroidetes

were

rarely

(13.9%)

and

(37.9%) and affiliated

with

454

In total, 172 genera were identified, although only seven were predominant at

455

relative abundances of more than 4%. The sum of the sequence number belonging to

456

these seven genera accounted for 84.7% of the total sequences. As shown in Figure

457

6(c), the dominant genera (in order) were Paracoccus (26.1%), Azoarcus (24.8%),


Page 23 of 40


458

Acetoanaerobium

459

Geosporobacter (4.6%) and Methanobacterium (4.3%).

(11.4%),

Labrenzia

(7.4%),

Dysgonomonas

(6.0%),

460

The genus Paracoccus was the most abundant in this hydrogenotrophic

461

denitrifying culture, and is considered as metabolically versatile bacterium with

462

bioenergetic flexibility.41 Most of the species of Paracoccus use nitrate as an

463

alternative electron acceptor to oxygen with nitrogen gas as final reduction product. A

464

range of organic and inorganic compounds including hydrogen can be used as

465

electron donors by the Paracoccus species, which are frequently observed in

466

bioreactors that show nitrate reduction with hydrogen.42,43 In addition, Vasiliadou et al.

467

used a pure culture of Paracoccus sp. to investigate the hydrogenotrophic

468

denitrification process, and nitrite accumulation was not observed throughout the

469

batch test.16

470

The genus Azoarcus was another dominant genus with a relative abundance close

471

to that of Paracoccus. Species of this genus have been reported as denitrifying

472

bacteria,44and are regarded as important groups that play a role in denitrification in

473

sewage treatment.45

474

The genus Acetoanaerobium, which contains the single species Acetoanaerobium

475

noterae, is an anaerobic bacterium that forms acetate from H2 and CO2.46 This

476

bacteria was frequently found in microbial fuel cells (MFCs).47This is the first report

477

that bacteria affiliated with Acetoanaerobium are enriched in the hydrogenotrophic

478

denitrifying community. The production of acetate could conceivably serve as an



479

electron donor and carbon source for the heterotrophic denitrification pathway that

480

might have occurred in this study. In the bioreactor, there are plenty of hydrogen as

481

electron donor and sodium bicarbonate as carbon source for hydrogenotrophic

482

denitrifier. The sodium bicarbonate could also serve as electron acceptor for

483

Acetoanaerobium to generate acetate and for Methanobacterium to generate methane.

484

Although the pathway of ecological interactions in the microbial community and the

485

contribution proportion of nitrogen removal are unavailable in this study, it can be

486

verified that there are a variety of metabolic types in such an ecological system with

487

hydrogen and inorganic carbon.

488

The genera Labrenzia, Dysgonomonas and Geosporobacter have never been

489

reported in hydrogenotrophic denitrifying culture in previous studies. However, they

490

were enriched in the bioreactor of this study. For the mechanism of significant

491

enrichment of these genera, it needs further investigation. Furthermore, the genus

492

Methanobacterium, which is hydrogenotrophic methanogenic archaea, was also

493

observed, suggesting that methanogenesis might also occur in this system.

494

Previous reports on the microbial community structure of hydrogenotrophic

495

denitrification are rare. Researchers have used denaturing gradient gel electrophoresis

496

and phylogenetic analysis to identify the dominant groups in hydrogenotrophic

497

denitrification cultures.23,48 In addition, studies have created clone libraries of the 16S

498

rRNA gene to reveal the microbial community structures.49 However, the information

499

obtained through these DNA fingerprinting analysis was too limited to reflect the


Page 24 of 40

Page 25 of 40


500

completely microbial community, because of the finite quantity of clones. In order to

501

completely reflect the microbial community, Mao et al. applied high-throughput

502

sequencing to characterize microbial communities of enriched hydrogenotrophic

503

denitrifying cultures.42 In their study, the most dominant populations observed were

504

identified as Thauera species, which had a relative abundance of nealy 60%. Chen et

505

al. analyzed the microbial community of biofilm from a bio-ceramsite reactor of

506

hydrogenotrophic denitrifying through 454-pyrosequencing.

507

genus was found to be Acinetobacter when the bioreactor was operated at pH of 7.0,

508

while genus Planomicrobium was most dominant at pH of 9.0. Moreover, the genus

509

Thauera and Paracoccus were also observed in the bio-ceramsite reactor. Zhang et al.

510

analyzed the microbial community of a membrane-biofilm reactors (MBfR) of

511

hydrogenotrophic denitrifying through 454-pyrosequencing.51 The most abundant

512

genera in the MBfR feeding nitrate as the sole electron acceptor was found to be

513

denitrifying bacteria, including Stenotrophomonas and Dechloromonas.

50

The most dominant

514

3.7 Implications to the environmental application

515

The performances of hydrogenotrophic denitrification and characteristics of N2O

516

accumulation were investigated through a series of batch tests under various

517

environmental conditions. It has been confirmed that nitrate could be efficiently

518

removed through hydrogenotrophic denitrification under the general condition in

519

practical engineering, including the pH, temperature, inorganic carbon content and

520

hydrogen concentration. Among the batch tests, the N2O was only observed to



Page 26 of 40

521

massively accumulated under slightly acidic condition and temperature dropping to

522

15 °C. By contrast, the N2O accumulation was extremely low under other test

523

conditions that were common in practical engineering. This result indicated that

524

hydrogenotrophic denitrification is an adaptive and promising technology. Its

525

advantages are not only the efficient nitrogen removal performance but also the

526

mitigation potential of N2O emission. Although it was observed that the N2O

527

massively accumulated only under some specific conditions, the implications of this

528

finding was still significant for practice, considering there would be some nitrogen

529

removal requirement under such condition in engineering. For instance, in the winter

530

of cold region, the temperature of nitrate-polluted water may drop below 15 °C.

531

some region, the pH of nitrate-polluted water may be lower than 7.0.

532

results in this study, if the hydrogenotrophic denitrification were used in such

533

condition, the N2O emission would be concerned and mitigation measures should be

534

considered.

52

10

In

According to

535

The microbial community of hydrogenotrophic denitrification was revealed by

536

high throughput sequencing. The dominant species were found to belong to the genera

537

Paracoccus (26.1%), Azoarcus (24.8%), Acetoanaerobium (11.4%), Labrenzia (7.4%),

538

and Dysgonomonas (6.0%). These findings could contribute to the knowledge of the

539

hydrogenotrophic denitrifiers.

540

Acknowledgements

541

This work was financially supported by the National Water Pollution Control and

542

Treatment Science and Technology Major Project of China (No.2012ZX07205-001)


Page 27 of 40


543

and the China Postdoctoral Science Foundation (No.2014M560982).

544

Supporting Information Available

545

Relevant information on batch tests condition, nitrate reduction rate, maximal

546

NO2--N and N2O-N accumulation ratio; methods of PCR amplification, purification

547

and re-amplification; Rarefaction curve plotting OTUs numbers. This information is

548

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

549

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(a) Phylum

0.4% 4.4%

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0.60% 0.70% 4.37% 6.66%

(b) Class

Betaproteobacteria

Proteobacteria 13.9%

Firmicutes

Clostridia

7.09% 37.89%

Bacteroidetes 17.4%

Euryarchaeota

Alphaproteobacteria

Bacteroidia Flavobacteria

17.25%

64.0%

Methanobacteria

Others

Gammaproteobacteria 25.44%

Others

(c) Genus 3.83% 0.57% 0.66% 8.08% 1.06% 1.09% 4.34%

26.11%

4.61% 5.97% 7.44% 11.40%

24.84%

Paracoccus Azoarcus Acetoanaerobium Labrenzia Dysgonomonas Geosporobacter Methanobacterium Proteiniphilum Dethiobacter Stappia Aquamicrobium Others Unclassified

Figure 6. The relative abundances of major bacterial groups at (a) Phylum, (b) Class and (c) Genus levels (sequence percentage >0.5%).


Nitrogen Removal and N2O Accumulation during Hydrogenotrophic

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