Dissimilatory Nitrate Reduction Processes in Typical Chinese Paddy

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Dissimilatory nitrate reduction processes in typical Chinese paddy soils: rates, relative contributions and influencing factors Jun Shan, Xu Zhao, Rong Sheng, Yongqiu Xia, Chaopu Ti, Xiaofei Quan, Shuwei Wang, Wenxue Wei, and Xiaoyuan Yan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01765 • Publication Date (Web): 06 Aug 2016 Downloaded from http://pubs.acs.org on August 7, 2016

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Environmental Science & Technology

Cover Page June 14th, 2016

Date of preparation: Number of text pages:

34

Number of figures:

4

Numbers of tables:

3

Type of paper:

Research Paper

Word count = 6836 (4736 text + 4 figures + 3 tables)

Title: Dissimilatory nitrate reduction processes in typical Chinese paddy soils: rates, relative contributions and influencing factors Authors: Jun Shan1, Xu Zhao1, Rong Sheng2, Yongqiu Xia1, Chaopu ti1, Xiaofei Quan1,3, Shuwei Wang1,3, Wenxue Wei2, Xiaoyuan Yan1* Address: 1

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China

2

Key Laboratory of Agro-ecological Processes in Subtropical Regions and Taoyuan Agro-ecosystem Research Station, Soil Molecular Ecology Section, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China

3

University of Chinese Academy of Sciences, Beijing 100049, China

*: Corresponding Author email: [email protected]; Tel: +86-025 8688 1530 Fax: +86-025 8688 1000

1

ACS Paragon Plus Environment


1

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Abstract: Using soil slurry-based 15N tracer combined with N2/Ar technique, the potential

2 3

rates

of

denitrification,

anaerobic

ammonium

oxidation

(anammox)

and

4

dissimilatory nitrate reduction to ammonium (DNRA), and their respective

5

contributions to total nitrate reduction were investigated in 11 typical paddy soils

6

across China. The measured rates of denitrification, anammox and DNRA varied

7

from 2.37 to 8.31 nmol N g-1 h-1, 0.15 to 0.77 nmol N g-1 h-1 and 0.03 to 0.54 nmol

8

N g-1 h-1, respectively. The denitrification and anammox rates were significantly

9

correlated with the soil organic carbon content, nitrate concentration, and the

10

abundance of nosZ genes. The DNRA rates were significantly correlated with the

11

soil C/N, extractable organic carbon (EOC)/NO3– ratio, and sulfate concentration.

12

Denitrification was the dominant pathway (76.75–92.47%), and anammox (4.48–

13

9.32%) and DNRA (0.54–17.65%) also contributed substantially to total nitrate

14

reduction. The N loss or N conservation attributed to anammox and DNRA was

15

4.06–21.24 and 0.89–15.01 g N m-2 y-1, respectively. This study reports the first

16

simultaneous investigation of the dissimilatory nitrate reduction processes in paddy

17

soils, highlighting anammox and DNRA play important roles in removing nitrate

18

and should be considered when evaluating N transformation processes in paddy

19

fields.

20

Key word: Denitrification; Anammox; DNRA; 15N tracer; N2/Ar technique

2


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21

1. Introduction

22

Nitrogen (N) is generally the key element limiting rice production; thus, there

23

has been much research on the factors that affect N retention and loss in paddy

24

fields1, 2. The waterlogged conditions during the main stage of rice growth provide a

25

unique environment for dissimilatory nitrate reduction processes including

26

denitrification, anaerobic ammonium oxidation (anammox) and dissimilatory nitrate

27

reduction to ammonium (DNRA)2, 3. Among these processes, denitrification has

28

been intensively studied4, 5 and was considered to be the major pathway converting

29

fixed N to N2 until the discovery of anammox, in which ammonium is oxidized to

30

dinitrogen using nitrite as an electron acceptor under anaerobic conditions6-8. Much

31

of what is known on anammox comes from aquatic environments, including marine

32

sediments9, river estuaries10-12, and freshwater and lake sediments13,

33

anammox significantly contributes to N loss (up to 79% of the total N2 production)15.

34

Studies on the occurrence and role of anammox in terrestrial ecosystems are limited,

35

although diverse anammox bacteria have been found in agricultural soils16. Until

36

now, few studies have reported anammox activity and estimated the importance of

37

anammox in N loss in paddy and farm soils, where anammox accounts for 0.4–37%

38

of the total N2 production depending on the soil type and profile17-23.

14

, where

39

In addition to anammox, DNRA has also been recognized in soil and may act

40

as the dominant pathway for the removal of nitrate under specific conditions (e.g.,

41

low redox potential and high ratio of electron donor to electron acceptor)24, 25. Only

42

three studies have reported DNRA rates in paddy soils, showing that DNRA

3



43

contributed 3.9–25.4% of total nitrate consumption, depending on the abundance of

44

DNRA bacteria and the environmental conditions26-28. It is possible that DNRA is

45

coupled with anammox by providing ammonium29. By converting nitrate to N2, both

46

denitrification and anammox result in a loss of N, whereas DNRA provides

47

ammonium for rice uptake and microbial immobilization, leading to N retention in

48

paddy soils.

49

In a single habitat, competition among denitrification, anammox and DNRA is

50

expected; however, previous studies related to dissimilatory nitrate reduction

51

processes in paddy soils focused on one or two independent processes (e.g.,

52

denitrification and anammox or DNRA)18, 20, 26. Simultaneous investigation of the

53

rates of denitrification, anammox and DNRA, and their relative contributions to total

54

nitrate reduction is lacking. In marine and estuarine ecosystems, the environmental

55

controls governing the competition among different dissimilatory nitrate reduction

56

processes include nitrate concentration, the availability of organic matter,

57

temperature and the sulfide concentration24,

58

denitrification, anammox and DNRA, and their relative contributions to

59

dissimilatory nitrate reduction process in paddy soils are unknown. Soil microbial

60

communities related to nitrate reduction play a crucial role in controlling rates of

61

denitrification, anammox, and DNRA, because these processes are mainly mediated

62

by microbial activities31. Microbial nitrate reduction functional genes, such as narG,

63

nirS/nirK, norB, nosZ, and hzsB are commonly used as molecular markers to

64

characterize the denitrifying and anammox bacteria community and to indirectly

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30

. However, the factors regulating

4


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65

reflect the activities of dissimilatory reduction processes32-34. Despite the potential

66

importance of the denitrifying microbial community to the rates and products of the

67

dissimilatory reduction processes, no clear linkages have been found between the

68

community composition or the functional gene abundance and the rates of the

69

dissimilatory reduction process31. Previous studies have shown that anammox rates

70

were significantly correlated with the abundance of hzsB genes20, 21, but little is

71

known about the relationships between denitrification or DNRA rates and the

72

microbial nitrate reduction functional genes in paddy soils.

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Laboratory slurry-based

15

N tracer techniques are often used as standard

74

methods to determine the potential rates of denitrification, anammox and DNRA

75

following the addition of either

76

sediments and soils35-37. In order to achieve high levels of

77

addition of

78

availability of N and may lead to an overestimation of dissimilatory nitrate reduction

79

rates especially in N-limited systems38. In addition, direct N2 flux measurement can

80

be precisely achieved by membrane inlet mass spectrometry (MIMS) and the N2/Ar

81

technique39, which is a relatively benign method without the addition of

82

substrates, although it cannot distinguish N2 production from specific processes,

83

such as denitrification and anammox39. Together with the intact soil/sediment core

84

incubation method, this technique has been successfully applied to quantify the in

85

situ N removal rates of submerged ecosystems (i.e., the denitrification rates

86

therein)41-43. However, no comparison has been made between the laboratory

15

NO3– or

15

15

NO3– alone or in combination with 15

15

NH4+ to the

N enrichment, the

NH4+ is often excessive, which consequently increases the

15

N

5



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slurry-based 15N tracer technique and the soil core incubation-based N2/Ar technique,

88

and whether the rates of denitrification and anammox from slurry-based 15N tracer

89

experiments can approximate the activities of the N removal rates without

90

15

N-substrate addition is unknown.

91

Hence, the objectives of the present study were to: i) simultaneously determine

92

the potential rates of denitrification, anammox and DNRA, and evaluate their

93

relative contributions to total nitrate reduction using a laboratory slurry-based

94

tracer technique, ii) elucidate the critical factors that influence the dissimilatory

95

nitrate reduction processes, and iii) assess whether the rates of denitrification and

96

anammox from slurry-based 15N tracer experiments can approximate the N removal

97

rates without utilizing 15N-substrate in typical paddy soils in China.

98

2. Materials and methods

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2.1 Soil sampling

15

N

100

Eleven paddy soil samples were collected across China (21°0`N to 127°11`N

101

and 102°45`E to 127°11`E) in July 2014 (See details in Table S1 of the supplemental

102

material). These soil samples were chosen because their locations could mainly

103

represent the dominant rice production areas in China. Urea was the routinely

104

chemical N fertilizer in these fields. For sites CS, LY, YX, JD, YA1 and YA2, 240–

105

300 kg urea-N ha-1 were applied, while for site HL, YT, ZJ, JMS, and LA, 120–180

106

kg urea-N ha-1 were applied in the rice season. All soil samples were collected at 0–

107

20 cm depth from field plots with at least three replicates (ca. 2–4 kg for each

108

subsample) for each location and were immediately transferred to sterile plastic bags

6


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with ice packs (4 °C) and were shipped to the laboratory as soon as possible. Each

110

soil sample was divided into three parts: one subsample was air-dried and sieved to

111

< 2 mm for physicochemical properties analysis, the second subsample was stored at

112

4 °C and was processed within 3 days for soil slurry and soil core incubation

113

experiments, and the third subsample was frozen at –80 °C for molecular analysis.

114

2.2 Analysis of soil characteristics

115

The total C and N contents of the soils were analyzed using an elemental

116

analyzer (Elementar Cario EL III; Elementar Analysensysteme GmbH, Germany).

117

The inorganic N (ammonium and nitrate) and sulfate in the soils were extracted with

118

2 M KCl and water, respectively, and were determined on a continuous flow

119

analyzer (SA1000; Skalar Analytical, Breda, the Netherlands). The soil organic

120

carbon (SOC) content was determined via potassium permanganate oxidation, and

121

the soil extractable organic carbon (EOC) content was determined by a total organic

122

carbon analyzer (TOC-VCS/CP, Shimadzu, Japan). Soil pH was measured using a

123

CaCl2 solution at a ratio (soil:CaCl2) of 1:2.5. The cation exchange capacity (CEC)

124

of the soils was determined using the ammonium acetate method. The soil texture

125

was analyzed using a Mastersizer 2000 (Malvern Instruments Ltd., Malvern, UK).

126

All soil parameters were analyzed in triplicate.

127

2.3 Measuring the denitrification, anammox and DNRA rates using the

128

technique

15

N tracer

129

The potential rates of denitrification and anammox were measured by soil

130

slurry incubation experiments using the 15N isotope pairing technique and the MIMS

7



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(Bay Instruments, Easton, MD) determination of 29N2 and 30N2 in the soil slurry10, 30.

132

Briefly, soil slurries were prepared by mixing fresh soils and helium (He)-purged

133

water at a ratio (soil:water) of 1:5 in 12 mL glass vials (Exetainer; Labco, UK). The

134

vials were then transferred to a vertical shaker and pre-incubated for 5–8 days at

135

20 °C to eliminate residual nitrate and oxygen. Subsequently, all the vials were

136

divided into three groups, which were spiked with 100 µL He-purged stock solution

137

of

138

final concentration of 100 µM

139

performed at 20 °C and were stopped by adding 200 µL saturated HgCl2 solution at

140

time points of 0, 1, 3, 5, and 7 h, after which the abundance of 29N2 and 30N2 in the

141

vials was directly determined by MIMS13. The potential rates of anammox and

142

denitrification, and their relative contributions to the total N2 production were

143

estimated by the accumulation of 29N2 and 30N2 during the incubations amended with

144

15

145

Dalsgaard9. Briefly, both anammox and denitrification generated

146

respective contributions of each process to the total 29N2 production were quantified

147

by eq 1:

15

NH4+ (99.3%

15

N),

15

NH4+ plus 15

14

NO3– or

15

NO3– (99.2%

15

N), resulting in a

N in each vial. Soil slurry incubations were

NO3– according to the methods and equations described in Thamdrup and 29

N2; thus, the

148

= + (1)

149

where, P29, A29, and D29 (nmol N g-1 h-1) represent the total 29N2 production rate, the

150

29

151

denitrification, respectively. Because the

152

denitrification follow random isotope pairing, D29 can also be estimated by the

N2 production rate from anammox, and the 28

N2,

29

29

N2 production rate from

N2, and

30

N2 generated from

8


Page 9 of 34


153

following equation: = × 2 × (1 − ) × (2)

154

where P30 (nmol N g-1 h-1) is the total

155

fraction of

156

concentrations of nitrate before and after the addition of

157

potential rates of anammox and denitrification were estimated by the following

158

equations:

15

30

N2 production rate and Fn is the mole

N in the nitrate pool, which can be calculated by the measured 15

NO3–. Consequently, the

= + 2 × (3) = − (4) 159

where Dtotal and A29 (nmol N g-1 h-1) are the potential rates of denitrification and

160

anammox, respectively.

161

The potential rate of DNRA was measured using the OX/MIMS method

162

(15NH4+ oxidation technique and MIMS analysis) developed by Yin et al.35. Briefly,

163

soil slurries were prepared and pre-incubated as for the denitrification and anammox

164

experiments mentioned above. After 5–8 days’ pre-incubation, 50 µL of

165

stock solution was added to the slurries, resulting in a final concentration of 100 µM

166

15

167

immediately preserved with 100 µL saturated HgCl2 solution and were treated as the

168

initial samples. The remaining vials were further incubated for 24 h at 20 °C,

169

followed by the addition of 100 µL saturated HgCl2 solution and were treated as the

170

final samples. All slurry samples were stirred and purged with He for 30 min to

171

eliminate the

15

NO3–

N in each vial. At the beginning of the incubation, half of the vials were

29

N2 and

30

N2 produced by denitrification and anammox during the

9



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172

incubation. The slurry vials were then sealed, and 200 µL hypobromite iodine

173

solution was injected to oxidize the DNRA-produced 15NH4+ into 15N gas (29N2 and

174

30

175

were measured by MIMS35 and were used to calculate the concentration of

176

DNRA-produced

177

calculated by eq 5:

N2). The concentrations of the generated 15N gases in the initial and final samples

15

NH4+ during the incubation. The potential rate of DNRA was

RDNRA

178

(  =

15

NH+4 

Final

−  15 NH4+ 

W ×T

Initial

) ×V (5)

179

where RDNRA (nmol N g-1 h-1) is the measured DNRA rate, [15NH4+]Final and

180

[15NH4+]Initial (nmol N L-1) are the measured concentrations of 15NH4+ in the final and

181

initial samples of the soil slurries, respectively, V (L) is the volume of the vial, W (g)

182

is the dry weight of the soil, and T (h) is the incubation time.

183

2.4 Measuring the net N2 fluxes using the soil core incubation-based N2/Ar

184

technique

185

Soil cores were prepared by manually adding 600 g fresh soil to a PVC tube (8

186

cm inner diameter and 10 cm depth), with the bottom sealed with an air-tight

187

Plexiglas plate. The soil inside the PVC tube was then homogenized flooded with

188

1.1 L distilled water and capped on the top using seals with two sampling ports and a

189

magnetic stirrer suspended from the top. All the soil cores were then transferred into

190

a water bath container equipped with a carousel housing four rotating magnets and

191

incubated at 20 °C. The overlying water in the PVC tubes was gently mixed by a

192

magnetic stirrer driven by the internal rotating magnets to homogenize the

193

floodwater and to stimulate the water flow under field conditions. After 10


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194

pre-incubation for 12 h, the overlying water was periodically sampled to measure

195

the dissolved N2 concentrations in the overlying water by MIMS39, 41. The net N2

196

flux from each soil core was calculated based on the linear regression of the N2

197

change in the overlying water during incubation. For each soil, three replicate soil

198

cores were performed.

199

2.5 DNA extraction and quantitative PCR (q-PCR)

200

DNA was extracted from 0.5 g fresh soil using a FastDNA spin kit for soil (MP

201

Biomedicals, Cleveland, OH, USA) according to the manufacturer’s instructions.

202

The quantity and quality of the extracted DNA were determined using a NanoDrop

203

spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Real-time

204

q-PCR analysis was performed with the extracted DNA to determine the abundance

205

of the bacterial 16S rRNA gene, denitrification functional genes (narG, nirS/nirK

206

and nosZ) and the anammox functional gene (hzsB). The primers were 1369F/1492R

207

for the bacterial 16S rRNA44, 571F/773R for narG45, Cd3aF/R3cd for nirS46,

208

876F/1055R for nirK45, 1126F/1381R for nosZ45, and HSBeta396F-HSBeta742R for

209

hzsB20. The gene copy numbers of the 16S rRNA, narG, nirS/nirK, nosZ and hzsB

210

were determined in triplicate using an ABI 7900 sequence detection system and the

211

thermal cycling conditions for the 16S rRNA gene, denitrification genes and

212

anammox gene can be found in Sheng, et al.47, Chen et al.45 and Yang et al.20,

213

respectively. The standard curves for the 16S rRNA, narG, nirS/nirK, nosZ and hzsB

214

genes were created using a 10-fold dilution series (102–109 copies) of the standard

215

plasmids DNA.

11



216

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2.6 Statistical analysis

217

Pearson correlation analysis was performed to evaluate the correlations among

218

the N transformation rates (net N2 flux, denitrification, anammox and DNRA), soil

219

physicochemical properties and microbial functional genes using the SPSS 16.0

220

package. One-way analysis of variance (ANOVA) followed by Fisher’s least

221

significant difference (LSD) test was performed to evaluate the differences among

222

means of different N transformation rates, and the significance level was set at P

Dissimilatory Nitrate Reduction Processes in Typical Chinese Paddy

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