The abundance and community structure of active ammonia-oxidizing archaea and ammonia-oxidizing bacteria shape their activities and contributions in coastal wetlands

Journal Pre-proof The abundance and community structure of active ammonia-oxidizing archaea and ammonia-oxidizing bacteria shape their activities and contributions in coastal wetlands Chen Wang, Shuangyu Tang, Xiangjun He, Guodong Ji PII:

S0043-1354(19)31241-2

DOI:

https://doi.org/10.1016/j.watres.2019.115464

Reference:

WR 115464

To appear in:

Water Research

Received Date: 18 October 2019 Revised Date:

19 December 2019

Accepted Date: 31 December 2019

Please cite this article as: Wang, C., Tang, S., He, X., Ji, G., The abundance and community structure of active ammonia-oxidizing archaea and ammonia-oxidizing bacteria shape their activities and contributions in coastal wetlands, Water Research (2020), doi: https://doi.org/10.1016/ j.watres.2019.115464. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical Abstract. The 13C labelled DNA was obtained after isotopic incubation and DNA-SIP selection. High-throughput sequencing technology was used to analyse microbial community structure to evaluate cell-specific activity for AOA and AOB. Quantitative PCR was used to measure gene abundance to calculate cell numbers for AOA and AOB. Finally, AOA and AOB activity were provided in four coastal wetlands.

1

The abundance and community structure of active

2

ammonia-oxidizing archaea and ammonia-oxidizing bacteria

3

shape their activities and contributions in coastal wetlands

4

Chen Wang, Shuangyu Tang, Xiangjun He, Guodong Ji*

5 6

Key Laboratory of Water and Sediment Sciences, Ministry of Education, Department

7

of Environmental Engineering, Peking University, Beijing 100871, China

8

*Corresponding author. E-mail address: [email protected]

9

10

Abstract

11

Aerobic ammonia oxidation, an important part of the global nitrogen cycle, is

12

thought to be jointly driven by ammonia-oxidizing bacteria (AOB) and

13

ammonia-oxidizing archaea (AOA) in coastal wetlands. However, the activities and

14

contributions of AOA and AOB in coastal wetlands have remained largely unknown.

15

Here, we investigated the oxidation capability of AOA and AOB in four types of

16

typical coastal wetlands (paddy, estuary, shallow and reed wetland) in the Bohai

17

region in China using DNA-based stable-isotope probing (DNA-SIP), quantitative

18

PCR and high-throughput sequencing techniques. We found that the community

19

structure of AOB varied substantially, and the AOA structure was more stable across

20

different coastal wetlands. The rate of AOA was 0.12, 0.84, 0.45 and 0.93 µg N g-1

21

soil d-1 in paddy, estuary, shallow and reed wetlands, and the rate of AOB was 5.61,

22

10.72, 0.74 and 1.16 µg N g-1 soil d-1, respectively. We found that the contribution of

23

AOA gradually increased from paddy to estuary to shallow wetland and finally to reed

24

wetland, with values of 2.03%, 7.25%, 37.53% and 44.51%, respectively. Our results

25

provide new insight into the mechanisms of the differences in activities and the

26

contributions of AOA and AOB in different coastal wetlands, and our findings may

27

contribute to further understanding of the global nitrogen cycle.

28 29 30 31 32 33 34

35

Introduction

36

Serving as buffer zones between the marine zones and inland regions, coastal

37

wetlands are among the regions most vulnerable and sensitive to global environment

38

change (Yu et al. 2016). The differences in land-use form various microbial patterns,

39

which further change the biogeochemical cycle in coastal wetlands. The nitrogen

40

cycle is one of the most important element cycles in coastal wetlands. Around the

41

world, coastal wetlands play a significant role as a buffer zone between inland and

42

marine ecosystems in the nitrogen cycle. As the first and rate-limiting step of

43

nitrification, ammonia oxidation is a key process in the global nitrogen cycle

44

(Pratscher et al. 2011). To date, ammonia oxidizing archaea (AOA) and bacteria

45

(AOB) have been considered to be jointly responsible for the ammonia oxidation

46

process (JC et al. 2004; Könneke et al. 2005; Hatzenpichler et al. 2008; de la Torre et

47

al. 2008; Jia and Conrad 2009). AOA and AOB have been reported to present different

48

abundance, community structure and activity patterns in different biotopes. However,

49

the mechanisms for the differences in the activities and contributions of AOA and

50

AOB in coastal wetlands is still unclear.

51

Generally, it has been proposed that AOA contributes more to ammonia

52

oxidation in wetlands than AOB, for AOA was often reported to be outnumber by

53

AOB by about an order of magnitude in various types of wetlands (Treusch et al.

54

2005; Leininger et al. 2006; Shen et al. 2008; Yarwood et al. 2013). At the same time,

55

the half-saturation constant for ammonium for some cultured AOA strains

56

(Martens-Habbena et al. 2009; Jung et al. 2011; Kim et al. 2012) was much lower

57

than AOB strains, which revealed that AOA had a greater affinity for substrates and

58

had competitive advantages over AOB, particularly in oligotrophic environments

59

(Verhamme et al. 2011). However, the abundance of the amoA gene, transcript or

60

protein was not sufficient to explain ammonia oxidation activity (Pester et al. 2011).

61

As AOA had a much lower growth rate and lower cell-specific ammonia oxidation

62

activity compared to AOB (BELSER 1979; Jiang and Bakken 1999; Tourna et al.

63

2011), AOA was thought to be less significant than AOB, although it possessed a

64

numerically dominant abundance. A better approach may be to evaluate the

65

contribution of AOA and AOB from the perspectives of both abundance and

66

community structure (which could reflect the variance of cell-specific ammonia

67

oxidation activity). In a previous study, cell-specific activity and gene abundance were

68

used to reveal the contribution of AOA and AOB (Schauss et al. 2009). However, the

69

researchers used several groups of cell-specific activity data without rigorous

70

consideration of the microbial community structure, which made the results

71

unreliable.

72

Several studies measured and compared the nitrification potentials of AOA and

73

AOB through the method of inhibitor experiments, in which acetylene, 1-octyne,

74

allylthiourea (ATU), sulfadiazine, dicyandiamide and antibiotics have been used as

75

inhibitors. Some antibiotics showed little inhibitory effect on AOA while a strong

76

inhibitory effect on AOB, which make them be exploited to distinguish the activities

77

of AOA and AOB (Schauss et al. 2009; Lehtovirta-Morley et al. 2011; Shen et al.

78

2013; Zheng et al. 2014). However, this method could only provide a rough

79

recognition of AOA and AOB activity and could not explain the variance in the

80

activity of AOA and AOB. Moreover, research regarding AOA enrichment proposed

81

that it was not sufficient to measure the activity of AOA and AOB using inhibitors

82

(Jung et al. 2011; Zhalnina et al. 2014). The results that AOA was also inhibited or

83

that AOB was not completely inhibited suggested that the method of using inhibitors

84

was not robust. More advanced DNA stable-isotope probe (DNA-SIP) methods have

85

been applied to investigate active archaeal and bacterial ammonia oxidizers (Jia and

86

Conrad 2009; Zhang et al. 2010; Wang et al. 2014). Both AOA and AOB have been

87

indirectly shown to be active and dominant in ammonia oxidation in different studies

88

through a single label of the archaeal or bacterial amoA gene. However, in most cases,

89

both archaeal and bacterial amoA genes were labeled, and this result led to a

90

continued debate about how to use active microbial abundance to evaluate activities

91

(Weiwei et al. 2011). To date, the activities of AOA and AOB remain controversial.

92

DNA-SIP was effective for distinguishing active ammonia oxidizers while

93

high-throughput sequencing technology could provide a clear recognition of active

94

AOA and AOB microbial structures. The active cell numbers and weighted

95

cell-specific activity for AOA and AOB could be reflected by active gene abundance

96

and microbial community structure, respectively. On the basis of this assumption, the

97

archaeal and bacterial ammonia oxidation capabilities could be synthetically assessed.

98

In this study, we aimed to investigate the activities and contributions of AOA and

99

AOB in different coastal wetlands. Paddy fields (PF), estuary wetlands (EW), shallow

100

wetlands (SW) and reed wetlands (RW) in the Bohai region in China were chosen to

101

perform laboratory isotopic incubation. Our results first revealed the mechanism for

102

the difference in activities and contributions of AOA and AOB in various coastal

103

wetlands based on the abundance and community structure of active AOA and AOB,

104

which may provide new insights about the role of AOA and AOB in the global

105

nitrogen cycle.

106

METHODS

107

Sampling and characteristics measurement

108

Sampling was conducted in July 2012. Soil/sediment samples were collected

109

from four types of wetlands along the coastline of the Bohai rim (34°23’-43°29’N and

110

113°23’-125°50’E, Fig. S1), namely, in paddies, estuary wetlands, reed wetlands and

111

shallow wetlands. At each sampling site, five individual soil/sediment cores were

112

randomly collected from the surface layer with a 30 cm depth and within a 10 × 10 m

113

area and then composited into a single sample for each site. The samples were air

114

dried and then sieved to 2 mm before analysis.

115

Soil/sediment pH was determined in a 1:2.5 soil/water suspension. Ammonium

116

(NH4+), nitrate (NO3−) and nitrite (NO2−) were extracted with 2 M KCl and were

117

measured using a spectrophotometer (UV-1800, SHIMADZU, Japan). Organic carbon

118

was determined with an elemental analyser (2400II CHNS/O, PerkinElmer, USA).

119

Potential ammonia oxidation activity measured by inhibitors

120

The ammonia oxidation rate for AOA and AOB was measured with three

121

replicates in two sets of experiments (groups A and B), which was designed based on

122

a described previously method (Zheng et al. 2014). The homogenized, field-moist

123

soil/sediment samples (10.0 g) were weighed into 150-mL incubation flasks, and 80

124

mL of solution (0.4 g/L MgCl2, 0.5 g/L KCl, 0.2 g/L KH2PO4, 1 g/L NaCl, 0.1 g/L

125

CaCl2, and 10 mM KClO3; Fisher Scientific) was added to each replicate. Group B

126

was combined with a final concentration of 100 mg/L penicillin to inhibit the activity

127

of bacteria. After preincubation for one day, a final concentration of 0.5 M ammonium

128

chloride was added to all groups. The flasks were incubated at 30 °C, and the mud

129

was sampled after 24-hour incubation to define the total ammonia oxidation rate

130

(group A) and the archaeal ammonia oxidation rate (group B) through analyses of

131

nitrite concentration changes. The bacterial ammonia oxidation rate was determined

132

by subtracting group B from group A.

133

Microcosm incubation and stable-isotope probing of active ammonia oxidizers

134

The DNA-SIP microcosms were constructed in 120-mL serum bottles with 20 g

135

samples and a 50 mL NaHCO3 solution sealed with rubber stoppers and plastic caps.

136

The air in the headspace of each bottle was replaced with synthetic air (80% N2 and

137

20% O2) to remove CO2 from the microcosms, and external bicarbonates were added

138

to the microcosms as the additional inorganic carbon (IC) sources. For each sample,

139

two groups of microcosms were supplemented, respectively, by two IC sources: one

140

was 100 mg L-1 NaH12CO3 and 5%

141

and 5%

142

performed in triplicate microcosms and incubated at 30 °C in the dark. The sodium

143

bicarbonates were added into the corresponding microcosms every week to maintain a

144

suitable concentration. After 56 days of cultivation, soils were sampled and then

145

immediately frozen at −80 °C for subsequent molecular analysis. The remainder of the

146

soil sample was used for the determination of potential nitrifying activity.

13

12

CO2, and the other was 100 mg L-1 NaH13CO3

CO2 (Sigma-Aldrich Co., St. Louis, MO, USA). All treatments were

147

DNA was extracted from the soil samples using a Fast Soil DNA Kit D5625-01

148

(Omega, USA) according to the manufacturer’s instructions. The concentration and

149

purity of extracted soil DNA was determined with a NanoDrop 2000 UV–Vis

150

spectrophotometer (Thermo Fisher, Wilmington, MA, USA). SIP gradient

151

fractionation was performed as previously described (Weiwei et al. 2011). For each

152

treatment, approximately 3 µg of the total DNA extracted from the incubated soils

153

was mixed with a CsCl stock solution to achieve an initial CsCl buoyant density of

154

1.725 g mL-1 to separate

155

centrifugation

156

ultracentrifugation tube in a Vti65.2 vertical rotor (Beckman Coulter Inc., Palo Alto,

157

CA, USA). The

158

ultracentrifugation at 177 000 x g for 44 h at 20 °C. DNA fractions were obtained by

159

displacing the gradient medium with sterile water from the top of the ultracentrifuge

was

13

12

C- and

performed

13

C-enriched DNA. The isopycnic density

using

a

5.1-mL

Quick-Seal

polyallomer

C-labeled DNA was separated from the original DNA by

160

tube using a syringe pump (New Era Pump Systems Inc., Farmingdale, NY, USA).

161

Approximately 14 DNA gradient fractions were obtained with equal volumes of

162

approximately 380 µL, and the refractive index of each fraction was measured using

163

an AR200 digital hand-held refractometer (Reichert Inc., Buffalo, NY, USA). The

164

fractionated DNA was purified and dissolved in 30 mL of TE buffer as previously

165

described (Freitag et al. 2006).

166

Quantitative PCR of amoA genes

167

Quantitative PCR (qPCR) was performed on a MyiQ2 Real-Time PCR Detection

168

System (Bio-Rad, USA) in 20 µL volume reaction mixtures containing the following

169

components: 10 µL SYBR Green I PCR master mix (Applied Biosystems, USA), 1

170

µL template DNA (sample DNA or plasmid DNA for standard curves), forward and

171

reverse primers, and sterile water (Millipore, USA). The protocol was set as

172

previously described (Chen et al. 2008).

173

High-throughput sequencing for the active amoA gene

174

The heavy DNA (fractions 2–6) obtained from isopycnic centrifugation of the

175

total DNA extracts was used for further sequencing analysis. Pyrosequencing of the

176

amoA

177

Arch-amoA26F/Arch-amoA417R (Park et al. 2010) and amoA-1F/amoA-2R

178

(Rotthauwe et al. 1997), respectively (Table S5), carried out by Majorbio (Shanghai,

179

China) using Illumina MiSeq sequencing. Chimera-free amoA gene sequences were

180

grouped into OTUs using a 97% similarity as a cut off, and the MOTHUR program

181

was used to generate the OTU-based Shannon diversity index and rarefaction

182

curve(Schloss et al. 2009). Phylogenetic analysis of the AOA or AOB community was

183

performed using MEGA6 software with the neighbor-joining and maximum

184

parsimony methods (Tamura et al. 2013). The raw sequence data have been deposited

gene

for

AOA

and

AOB

was

performed

using

the

primers

185

at the NCBI, with the accession number SRP237701.

186

Evaluation of AOA and AOB capability

187

The evaluation of AOA and AOB capability was carried out by a combination of

188

active microbial cell number and cell-specific activity. The active cell number was

189

defined by the microbial abundance, assuming that each AOB and AOA cell contained

190

2.5 and 1.0 of the amoA gene copy. The cell-specific activity was calculated as

191

follows:

=∑

192

×

×

193

where q stands for the weighted cell-specific activity of ammonia oxidizers, pi

194

stands for the cell-specific activity of i clusters that had been reported previously, ci

195

stands for the proportion of i clusters in the total microbial community, and ri stands

196

for the corrected coefficient for the i cluster under different soil conditions. The

197

corrected coefficient is summarized from previous studies (WATSON et al. 1971;

198

BELSER 1979; Stein and Arp 1998; Jiang and Bakken 1999) and presented in Table

199

S3.

200

Statistical analyses

201

Data processing and analysis were performed with SPSS Statistics 20 (IBM,

202

USA). A priori P-value of P < 0.05 was defined to test significant difference.

203

Results

204

Measured potential ammonia oxidation activity

205

The potential ammonia oxidation activity was assessed by measuring changes in

206

the nitrite concentration during incubation with penicillin. The measured total

207

ammonia oxidation activity was 5.93, 13.36, 1.16 and 3.24 µg N g-1 soil d-1 in PF, EW,

208

SW and RW, respectively (Fig. S6). The estuary wetland had the highest potential

209

ammonia oxidation activity compared with paddy, shallow and reed wetlands. The

210

measured activities and contributions of archaeal and bacterial ammonia oxidation

211

were also calculated. The measured contributions of AOA were 25.40%, 45.98%,

212

49.99% and 50.25% in PF, EW, SW and RW (Fig. S7), respectively.

213

Quantification of archaeal and bacterial amoA genes

214

The growth of AOA and AOB during the incubation period was determined by

215

the quantification of archaeal and bacterial amoA genes using quantitative PCR

216

(qPCR) at days 0 and 56. The copy numbers of the archaeal amoA genes increased by

217

4.84-, 4.32-, 9.91- and 40.2-fold in the 13C-incubated microorganisms of PF, EW, SW

218

and RW, respectively. Specifically, the copy numbers increased from 8.76×105 to

219

4.24×106, 6.30×106 to 2.72×107, 1.37×106 to 1.36×107 and 8.40×106 to 3.38×107

220

copies g-1 soil, respectively (Fig. S3). The copy numbers of the bacterial amoA genes

221

increased by 31.8-, 6.74-, and 13.6-fold in the

222

EW, and SW, respectively. Specifically, the copy numbers increased from 1.80×105 to

223

5.73×106, 9.93×105 to 6.69×106, and 1.71×105 to 2.32×106 copies g-1 soil, respectively

224

(Fig. S3). However, the copy numbers of bacterial amoA genes in RW decreased from

225

2.14×106 to 1.26×106 copies g-1 soil.

226

DNA-SIP analysis of AOA and AOB

13

C-incubated microorganisms of PF,

227

DNA-SIP was used to determine the active microbial functions responsible for

228

real ammonia oxidation. Ultracentrifugation of the total extracted DNA was

229

performed to separate 12C- and 13C-enriched DNA in four soils after incubation for 56

230

days. Real-time quantitative PCR of archaeal and bacterial amoA genes in different

231

fractions suggested strong labeling in four tested soils. In 13C-labeled control groups,

232

the highest abundance of both archaeal and bacterial amoA genes was detected in

233

heavy fractions with a buoyant density (approximately 1.735-1.755 g mL-1) higher

234

than that in the 12C-labeled control groups (approximately 1.725-1.730 g mL-1) (Fig.

235

S4), suggesting that AOA and AOB were well labeled through carbon assimilation

236

during their growth.

237

Furthermore, the genes obtained from heavy fractions represented active

238

microbes, which may be primarily responsible for ammonia oxidation (Pratscher et al.

239

2011). The abundance of the active amoA gene for AOA and AOB was summarized

240

from heavy fractions and is shown in Fig. 1. The ratios of gene copy numbers of

241

archaeal amoA genes in heavy DNA to all DNA fractions was 67.82%, 66.37%, 81.08%

242

and 68.22% in PF, EW, SW and RW, respectively. Meanwhile, the ratios for bacterial

243

amoA genes were 61.79%, 70.43%, 69.7% and 57.35%, respectively (Table 1).

244

According to previous studies, the cell numbers of active AOA and AOB could be

245

further calculated assuming that each bacterial and archaeal cell contains 2.5 and 1.0 of

246

the amoA gene copy. The cell numbers of active AOA were 2.88×106, 1.81×107,

247

1.10×107 and 2.30×107 per g soil in PF, EW, SW and RW, respectively. The cell

248

numbers of active AOB were 1.41×106, 1.88×106, 6.47×105 and 2.89×105 per g soil,

249

respectively.

250

Diversities of active AOA and AOB in different tested soils

251

The archaeal and bacterial amoA genes in the DNA samples of the heavy

252

fractions in 13C-treated microcosms were analyzed by pyrosequencing after 56 days of

253

incubation. The high-throughput sequencing of

254

understanding of the community structure for both active AOA and AOB. For AOA,

255

all sequences from tested soils could be grouped into three clusters: group 1.1a (which

256

could divided into 2 clusters upon detailed analysis) and group 1.1b (Fig. 2a). The

257

proportion of group 1.1a-1, represented by N. archaeon MY1, was 0.26% and 0.16%

258

in PF and RW. It was undetected in EW and SW. The proportion of group 1.1a-2,

13

C-amoA genes provided a clear

259

represented by N. maritimus SCM1, was only detected in EW, with a value of 4.34%.

260

The proportion of group 1.1b, represented by Nitrososphaera sp.JG1, was 99.74%,

261

95.66%, 100% and 99.84% in PF, EW, SW and RW, respectively. For active AOB, the

262

sequences could be grouped into one cluster of genus Nitrosomonas and four clusters

263

of Nitrosospira (Fig. 2b). The Nitrosomonas cluster accounted for 91.68% and 94.52%

264

of the labeled bacterial amoA genes in PF and RW. However, the proportion decreased

265

in EW and SW. The Nitrosomonas cluster and Nitrosospira multiformis cluster

266

became the two main clusters in EW, with values of 54.41% and 43.82%, respectively.

267

Meanwhile, the Nitrosospira multiformis cluster became dominant in SW, accounting

268

for 64.48% of total labeled bacterial amoA genes.

269

Evaluation of activities and contributions of AOA and AOB

270

The weighted cell-specific activity of AOA and AOB were determined by the

271

structure of active microbes depending on the measured AOA and AOB data. To make

272

the results more reliable, cell-specific activity was amended with certain fundamental

273

environmental parameters, mainly pH value and ammonia concentration. The

274

weighted cell-specific activity of AOA ranged narrowly from 2.88 to 3.31 femto mol

275

NH3 cell-1 d-1, while that of AOB ranged from 81.86 to 406.37 femto mol NH3 cell-1

276

d-1 (Fig. 3a). Combined with the active AOA and AOB cell numbers, the activity of

277

AOA and AOB was then calculated. The rate of AOA was 0.12, 0.84, 0.45 and 0.93

278

µg N g-1 soil d-1 in PF, EW, SW and RW, respectively, and the rate of AOB was 5.61,

279

10.72, 0.74 and 1.16 µg N g-1 soil d-1, respectively (Fig. 3b).

280

Discussion

281

DNA-SIP relied entirely on cell proliferation, thus providing new insight into

282

functional ammonia oxidizers using active microbial abundance instead of existing

283

abundance (Wang et al. 2014), which may be a more accurate means of assessing the

284

role of AOA and AOB. Previous studies determined the importance of AOA and AOB

285

based on existing gene abundance (Leininger et al. 2006; Chen et al. 2008). However,

286

that approach may be insufficient, as the microbial community may have no function

287

even with a relatively high abundance. In previous studies, AOA was confirmed to be

288

solely responsible for ammonia oxidation as there was no AOB labeled after isotopic

289

incubation (Zhang et al. 2010). In comparison, the role of AOB could be determined

290

in the same way if AOA was not labeled by isotope (Jia and Conrad 2009). In our

291

study, the successful labeling of the amoA gene by isotope demonstrates the function

292

of both AOA and AOB during incubation, which differs from previous studies. The

293

ratio of active genes to all gene abundance differed among the four biotopes, implying

294

a difference in the growth of AOA and AOB. Unfortunately, in the cases of labeling

295

both archaeal and bacterial amoA genes, the role of AOA and AOB could not be

296

determined conclusively.

297

The high-throughput pyrosequencing of both the archaeal and bacterial amoA

298

gene provided a detailed identification of active AOA and AOB in four coastal

299

wetlands. The main archaeal composition in the four coastal wetlands was group 1.1b

300

(with a composition greater than 95.66%), which has been demonstrated to be the

301

primary terrestrial group in the literature (Wang et al. 2014). The result that

302

amoA-containing archaeal populations were relatively constant was consistent with

303

previous studies (Jia and Conrad 2009). The stability of archaeal composition

304

reflected the stability of the archaeal community structure across various coastal

305

wetlands with different land-use types. The land-use types of coastal wetlands had

306

little impact on the community structure of archaeal ammonia oxidizers. In contrast,

307

the community structure of bacterial ammonia oxidizers varied in different biotopes.

308

The dominant AOA of PF was the Nitrosomonas cluster, which corresponded with

309

what has been reported in habitats with high ammonia concentrations.(Hastings et al.

310

1997; Taylor and Bottomley 2006). The proportion of Nitrosospira clusters increased

311

to active microcosms in EW and SW. Nitrosospira species have often been detected in

312

natural environments (Kowalchuk and Stephen 2001). However, the primary

313

proportion was the Nitrosomonas cluster in RW. The high variance in community

314

structure suggested a large response of bacterial ammonia oxidizers to various

315

land-use types. The distinct responses of AOA and AOB to land-use conversion (Liu

316

et al. 2017), nitrogen addition and urea amendment (Carey et al. 2016) supported our

317

results to some extent. For cell-specific activity, the AOB has a larger range than AOA

318

across coastal wetland types due to a larger range in community structure. The variety

319

of land-use types effected the average cell-specific activity of AOA to a small extent

320

but had a strong effect on AOB activity. The results may be determined by the

321

stability of the community structure of AOA in different coastal wetlands.

322

Based on the existing research that AOA had single amoA gene copies and AOB

323

had 2.5 copies per cell (Okano et al. 2004), the cell numbers for both AOA and AOB

324

were easily calculated in different wetlands. The cell numbers varied for both AOA

325

and AOB in four coastal wetlands, which implied that AOA and AOB may play

326

different roles in different wetlands. The ammonia oxidation rate was then calculated

327

by combining cell numbers and cell-specific activity. In addition, the potential

328

ammonia oxidation activity of the total ammonia oxidizers, AOA and AOB, was

329

measured using penicillin (which inhibited the activity of AOB) in order to present a

330

comparison with our evaluated results. The contributions of AOA in all wetlands were

331

lower than the results measured by penicillin. The higher results measured by

332

penicillin (Fig. S7) may be caused by the considerable expression abundance of the

333

AOB-amoA gene (Fig. S8). The activity and contribution of AOA would be

334

overestimated if AOB was not completely inhibited by penicillin. The highest

335

expression abundance of AOB-amoA implied that the contribution of AOA may be

336

largely overestimated in estuary wetlands. The community structure of AOB was also

337

investigated in detail based on RNA. The large proportion of Nitrosomonas and

338

Nitrosospira multiformis clusters (Fig. S8, Table S6), which had high cell-specific

339

activity, suggested that the role of AOB cannot be ignored and that the role of AOA

340

was largely overestimated, even when considering supplementation with penicillin.

341

However, our estimates of total ammonia oxidation rates exhibited the same trend as

342

results obtained from experiments (Fig. S6, Table S4), suggesting that our evaluation

343

was reliable.

344

The ammonia oxidation rates varied for both AOA and AOB in different

345

wetlands. However, the activities of AOA vary little across different land-use types

346

compared to AOB. The AOA activity was low in PF but significantly increased in EW,

347

SW and RW. This activity may be affected by the higher background ammonia

348

concentration (Fig. S5). Although the weighted cell-specific activity was extensive in

349

PF, the lower abundance led to a considerably lower ammonia oxidation rate for AOA,

350

which can be understood from the inhibition of a relatively higher ammonia

351

concentration and which had been previously demonstrated in both field surveys and

352

pure cultures (Tourna et al. 2011; Lehtovirta-Morley et al. 2011). The rate of AOA

353

increased by 6-, 2.75- and 6.75-fold in EW, SW and RW compared to PF. In such

354

samples with a lower ammonia concentration, AOA can survive in oligotrophic

355

conditions (Martens-Habbena et al. 2009; Verhamme et al. 2011), thus leading to an

356

increased archaeal ammonia oxidation rate. For AOB, the rate was much higher in PF

357

and EW and significantly decreased in SW and RW, which was primarily caused by

358

the lower ammonia affinity of AOB compared with AOA in low ammonia

359

concentration areas.

360

The contribution of AOA in ammonia oxidation gradually increased from PF to

361

EW to SW, and finally, to RW, with values of 2.03%, 7.25%, 37.53% and 44.51%

362

(Table S4), respectively. Similarly, the contribution of AOB was 97.97%, 92.75%,

363

62.47% and 55.49%, respectively. The value of the 2.03% proportion shows that AOA

364

contributes little in PF, whereas the rate of AOA was significantly lower than that of

365

AOB. Anthropogenic activities led to frequent fertilizer addition and higher ammonia

366

concentrations, which further illustrated the strong inhibition of AOA. Although the

367

rate of AOA increased by only 1.11-fold in RW and was even lower in SW when

368

compared with EW, the contribution was much higher, at approximately 44.51% and

369

37.53% for RW and SW, respectively, mainly because of the significant decrease in

370

the AOB rate. Many environmental factors have been reported to have an important

371

effect on AOA and AOB abundance and activity (Caffrey et al. 2007; Bouskill et al.

372

2012). AOB was concluded to be less resistant to oligotrophic conditions. Under these

373

conditions, AOA was more tolerant and in fact increased in activity. In favorable

374

environments, AOA tends to contribute more to ammonia oxidation.

375

Conclusion

376

In summary, we provide new insights into the mechanisms of variance in activity

377

and the contributions of AOA and AOB in different coastal wetlands based on active

378

abundance and community structure. Our results also suggested that the active

379

community structure of AOB varied more strongly among different biotopes, and the

380

active community structure of archaeal ammonia oxidizers was more stable.

381

Furthermore, our results also revealed that the contribution of AOA in ammonia

382

oxidation increased from PF to EW to SW, and finally to RW. AOA may be dominant

383

in coastal wetlands with less nitrogen or nutrient input where AOB was inhibited. The

384

results thus provide new insights into the mechanisms of variance in AOA and AOB

385

activity in different coastal wetlands, which may provide a basis for further

386

understanding of the global nitrogen cycle.

387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438

References: BELSER LW.1979.POPULATION ECOLOGY OF NITRIFYING BACTERIA. Annu Rev Microbiol 33: 309-333. Bouskill NJ, Eveillard D, Chien D, Jayakumar A, Ward BB. 2012. Environmental factors determining ammonia-oxidizing organism distribution and diversity in marine environments. Environ Microbiol 14: 714-729. Caffrey JM, Bano N, Kalanetra K, Hollibaugh JT .2007. Ammonia oxidation and ammonia-oxidizing bacteria and archaea from estuaries with differing histories of hypoxia. Isme J 1: 660-662. Carey CJ, Dove NC, Beman JM, Hart SC, Aronson EL .2016. Meta-analysis reveals ammonia-oxidizing bacteria respond more strongly to nitrogen addition than ammonia-oxidizing archaea. Soil Biol Biochem 99: 158-166. Chen X, Zhu Y, Xia Y, Shen J, He J .2008. Ammonia-oxidizing archaea: important players in paddy rhizosphere soil. Environ Microbiol 10: 1978-1987. de la Torre JR, Walker CB, Ingalls AE, Koenneke M, Stahl DA .2008. Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ Microbiol 10: 810-818. Freitag TE, Chang L, Prosser JI .2006. Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient. Environ Microbiol 8: 684-696. Hastings RC, Ceccherini MT, Miclaus N, Saunders JR, Bazzicalupo M, McCarthy AJ .1997. Direct molecular biological analysis of ammonia oxidising bacteria populations in cultivated soil plots treated with swine manure. Fems Microbiol Ecol 23: 45-54. Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A, Daims H, Wagner M .2008. A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring. P Natl Acad Sci Usa 105: 2134-2139. JC V, K R, JF H, AL H, D R, JA E, D W, I P, KE N, W N, DE F, S L, AH K, MW L, K N, O W, J P, J H, R P, H B, C P, YH R, HO S .2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304: 66-74. Jia Z, Conrad R .2009. Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil. Environ Microbiol 11: 1658-1671. Environ Microbiol 11: 1658-1671. Jiang QQ, Bakken LR .1999. Comparison of Nitrosospira strains isolated from terrestrial environments. Fems Microbiol Ecol 30: 171-186. Jung M, Park S, Min D, Kim J, Rijpstra WIC, Damste JSS, Kim G, Madsen EL, Rhee S .2011. Enrichment and Characterization of an Autotrophic Ammonia-Oxidizing Archaeon of Mesophilic Crenarchaeal Group I.1a from an Agricultural Soil. Appl Environ Microb 77: 8635-8647. Kim J, Jung M, Park S, Rijpstra WIC, Damste JSS, Madsen EL, Min D, Kim J, Kim G, Rhee S .2012. Cultivation of a highly enriched ammonia-oxidizing archaeon of thaumarchaeotal group I.1b from an agricultural soil. Environ Microbiol 14: 1528-1543. Könneke M, Bernhard AE, Jr DLT, Walker CB, Waterbury JB, Stahl DA .2005. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437: 543-546. Kowalchuk GA, Stephen JR .2001. Ammonia-oxidizing bacteria: A model for molecular microbial ecology. Annu Rev Microbiol 55: 485-529. Lehtovirta-Morley LE, Stoecker K, Vilcinskas A, Prosser JI, Nicol GW .2011. Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil. P Natl Acad Sci Usa 108: 15892-15897. Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Schuster SC, Schleper C .2006. Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442: 806-809. Liu H, Wu X, Wang Q, Wang S, Liu D, Liu G .2017. Responses of soil ammonia oxidation and ammonia-oxidizing communities to land-use conversion and fertilization in an acidic red soil of southern China. Eur J Soil Biol 80: 110-120.

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498

Martens-Habbena W, Berube PM, Urakawa H, Jr DLT, Stahl DA .2009. Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461: 976-979. Okano Y, Hristova KR, Leutenegger CM, Jackson LE, Denison RF, Gebreyesus B, Lebauer D, Scow KM .2004. Application of real-time PCR to study effects of ammonium on population size of ammonia-oxidizing bacteria in soil. Appl Environ Microb 70: 1008-1016. Park B, Park S, Yoon D, Schouten S, Damste JSS, Rhee S .2010. Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria. Appl Environ Microb 76: 7575-7587. Pester M, Schleper C, Wagner M .2011. The Thaumarchaeota: an emerging view of their phylogeny and ecophysiology. Curr Opin Microbiol 14: 300-306. Pratscher J, Dumont MG, Conrad R .2011. Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil. P Natl Acad Sci Usa 108: 4170-4175. Rotthauwe JH, Witzel KP, Liesack W .1997. The ammonia monooxygenase structural gene amoA as a functional marker: Molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microb 63: 4704-4712. Schauss K, Focks A, Leininger S, Kotzerke A, Heuer H, Thiele-Bruhn S, Sharma S, Wilke B, Matthies M, Smalla K, Munch JC, Amelung W, Kaupenjohann M, Schloter M, Schleper C .2009. Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils. Environ Microbiol 11: 446-456. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF .2009. Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities. Appl Environ Microb 75: 7537-7541. Shen JP, Zhang LM, Zhu YG, Zhang JB, He JZ .2008. Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam. Environ Microbiol 10: 1601-1611. Shen T, Stieglmeier M, Dai J, Urich T, Schleper C .2013. Responses of the terrestrial ammonia-oxidizing archaeon Ca. Nitrososphaera viennensis and the ammonia-oxidizing bacterium Nitrosospira multiformis to nitrification inhibitors. Fems Microbiol Lett 344: 121-129. Stein LY, Arp DJ .1998. Ammonium limitation results in the loss of ammonia-oxidizing activity in Nitrosomonas europaea. Appl Environ Microb 64: 1514-1521. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S .2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30: 2725-2729. Taylor AE, Bottomley PJ .2006. Nitrite production by Nitrosomonas europaea and Nitrosospira sp AV in soils at different solution concentrations of ammonium. Soil Biol Biochem 38: 828-836. Tourna M, Stieglmeier M, Spang A, Koenneke M, Schintlmeister A, Urich T, Engel M, Schloter M, Wagner M, Richter A, Schleper C .2011. Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. P Natl Acad Sci Usa 108: 8420-8425. Treusch AH, Leininger S, Kletzin A, Schuster SC, Klenk HP, Schleper C .2005. Novel genes for nitrite reductase and Amo‐related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environ Microbiol 7: 1985-1995. Verhamme DT, Prosser JI, Nicol GW .2011. Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms. Isme J 5: 1067-1071. Wang B, Zhao J, Guo Z, Ma J, Xu H, Jia Z .2014. Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils. Isme J 9: 1062-1075. WATSON SW, GRAHAM LB, REMSEN CC, VALOIS FW .1971. LOBULAR, AMMONIA-OXIDIZING BACTERIUM, NITROSOLOBUS-MULTIFORMIS NOV-GEN NOV-SP. ARCHIV FUR MIKROBIOLOGIE 76: 183. Weiwei X, Caixia Z, Xiaowei Z, Youzhi F, Jiahua W, Xiangui L, Jianguo Z, Zhengqin X, Jian X, Zucong C .2011. Autotrophic growth of nitrifying community in an agricultural soil. Isme J 5: 1226-1236. Yarwood S, Brewer E, Yarwood R, Lajtha K, Myrold D .2013. Soil Microbe Active Community Composition and Capability of Responding to Litter Addition after 12 Years of No Inputs. Appl Environ Microb 79: 1385-1392. Yu J, Zhan C, Li Y, Zhou D, Fu Y, Chu X, Xing Q, Han G, Wang G, Guan B, Wang Q .2016. Distribution of carbon, nitrogen and phosphorus in coastal wetland soil related land use in the Modern Yellow River Delta. Sci Rep-Uk 6. Zhalnina KV, Dias R, Leonard MT, de Quadros PD, Camargo FAO, Drew JC, Farmerie WG, Daroub SH, Triplett EW .2014. Genome Sequence of Candidatus Nitrososphaera evergladensis from Group I.1b Enriched from Everglades Soil Reveals Novel Genomic Features of the

499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514

Ammonia-Oxidizing Archaea. Plos One 9. Zhang L, Offre PR, He J, Verhamme DT, Nicol GW, Prosser JI .2010. Autotrophic ammonia oxidation by soil thaumarchaea. P Natl Acad Sci Usa 107: 17240-17245. Zheng, Y., Hou, L., Newell, S., Liu, M., Zhou, J., and Zhao, H .2014. Community Dynamics and Activity of Ammonia-Oxidizing Prokaryotes in Intertidal Sediments of the Yangtze Estuary. Appl Environ Microb 80: 408-419.

515

The National Natural Science Foundation of China (No. 51679001), the Foundation

516

for Innovative Research Groups of the National Natural Science Foundation of China

517

(No. 51721006), and the National Key Research and Development Project of China

518

(No. 2019YFC0609204) provided support for this study.

519

Author contributions

520

G.D.J. designed the research; C.W. and X.J.H. performed the research; G.D.J. and

521

C.W. analyzed the data; and all the authors wrote the paper.

522

Competing financial interests

523

The authors declare no competing financial interests.

Acknowledgements

524

Graphical Abstract. The 13C labeled DNA was obtained after isotopic incubation and

525

DNA-SIP selection. High-throughput sequencing technology was used to analyze

526

microbial community structure to evaluate cell-specific activity for AOA and AOB.

527

Quantitative PCR was used to measure gene abundance to calculate cell numbers for

528

AOA and AOB. Finally, AOA and AOB activity was provided for the four coastal

529

wetlands.

530

Fig. 1. Total and active abundance of archaeal (A) and bacterial (B) amoA genes

531

in different soils incubated with

532

abundance in total DNA extracted from microcosms, and the active data were

533

summarized from labeled fractions after centrifugation. The data are expressed as the

534

mean ± standard error (n=3).

535

Fig. 2. Phylogenetic analysis of the active archaeal (A) and active bacterial (B)

536

amoA in

537

represent paddy fields, estuary wetlands, shallow wetlands and reed wetlands,

538

respectively. The percentages in the following brackets represent the OTU

539

distributions in different wetlands.

540

Fig. 3. Weighted cell-specific ammonia oxidation rate (femto mol NH3 oxidized

541

cell-1 d-1) (A) and ammonia oxidation rate (µg N g-1 soil d-1) (B) for AOA and AOB

542

in paddy fields (a), estuary wetlands (b), shallow wetlands (c) and reed wetlands

543

(d). (A) Sector size represents the percentage of each cluster. The data in the central

544

circle represent the final weighted cell-specific rate evaluated by proportion and the

545

data of each cluster under given soil conditions. (B) The data in the sectors represent

546

the ammonia oxidation rates for AOA (green) and AOB (red), respectively. Sector size

547

represents the contribution percentages for AOA and AOB. The data in the central

548

circle represent the total ammonia oxidation rate.

13

13

C for 56 days. The total data represent the gene

C-labeled DNA after incubation for 56 days. PF, EW, SW and RW

1

Table 1. The contributions of AOA and AOB calculated in four tested soils.

Soils

Microbes

Copy number of genes (copies g-1) a

Ratios of gene copy numbers in heavy DNA to all DNA fractions (%)

Number of labelled cells (cell g-1) c

femto mol NH3 oxidized cell-1 d-1 d

NH3 oxidized by AOA or AOB -1 -1 (µg N g d )

Contribution of AOA or AOB (%)

b

Copy number of genes in heavy DNA (copies g-1)

67.82

2,876,924

2,876,924

2.88

0.12

2.03

AOA

4.24×106

AOB

5.73×10

6

61.79

3,540,567

1,416,227

282.80

5.61

97.97

AOA

2.72×107

66.37

18,079,188

18,079,188

3.31

0.84

7.25

AOB

6.69×10

6

70.43

4,711,767

1,884,707

406.37

10.72

92.75

AOA

1.36×107

81.08

11,043,096

11,043,096

2.88

0.45

37.53

AOB

2.32×10

6

69.70

1,617,040

646,816

81.86

0.74

62.47

AOA

3.38×107

68.22

23,044,716

23,044,716

2.88

0.93

44.51

PF

EW

SW

Measured total ammonia oxidation rate -1 -1 (µg N g d )

5.73

5.32

11.56

10.39

1.19

0.67

2.09 2.99 286.20 1.16 55.49 a represents the gene copy number of archaeal or bacterial amoA genes in the total DNA extracted from soil microcosms. b represents the ratio of gene copy numbers in the heavy DNA fraction (1.735-1.750 g mL-1) to the total numbers in all DNA gradient fractions. c represents the cell numbers of labelled AOA and AOB assuming that each AOB and AOA cell contains 2.5 and 1.0 of amoA gene copy. d represents the weighted cell-specific rate of ammonia oxidation by AOA or AOB, and it was calculated by the percentage of some known cultured AOA or AOB. The percentage was evaluated by the OTU numbers obtained from sequencing analysis by heavy DNA fraction (Fig. 3). RW

AOB

2 3 4 5 6

Evaluated total ammonia oxidation rate -1 -1 (µg N g d )

1.26×106

57.35

722,610

289,044

Highlights We used a new method based to DNA-SIP to evaluate the ammonia oxidizing activity of AOA and AOB. The results of new method were close to the results of the penicillin microcosm incubation. AOA dominant in coastal wetlands with less nitrogen or nutrient input where AOB was inhibited.

Declaration of Interest Statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Guodong Ji