Cold Temperature Effects on Long-Term Nitrogen Transformation

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Cold temperature effects on long-term nitrogen transformation pathway in a tidal flow constructed wetland Yunmeng Pang, Yan Zhang, Xingjun Yan, and Guodong Ji Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04002 • Publication Date (Web): 13 Oct 2015 Downloaded from http://pubs.acs.org on October 13, 2015

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Environmental Science & Technology

Cold temperature effects on long-term nitrogen transformation pathway in a tidal flow constructed wetland

Yunmeng Pang, Yan Zhang, Xingjun Yan, Guodong Ji* Key Laboratory of Water and Sediment Sciences, Ministry of Education, Department of Environmental Engineering, Peking University, Beijing 100871, China

*Corresponding author. Tel.: +86 10 62755914 87; fax: +86 10 62756526. E-mail address: [email protected]

Intended for: Environmental Science & Technology Type of Contribution: Research Article

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ABSTRACT: The present study investigated long-term treatment performance and

6

nitrogen transformation mechanisms in tidal flow constructed wetlands (TFCWs) under

7

4, 8 and 12 °C temperature regimes. High and stable ammonium (NH4+-N) removal

8

efficiency (93-96%) was achieved in our TFCWs, whereas nitrate (NO3--N) was

9

accumulated at different levels under different temperatures. Quantitative response

10

relationships showed anammox/amoA, (narG+napA)/amoA, and (narG+napA)/bacteria

11

were the respective key functional gene groups determining 4, 8, and 12 °C NO3--N

12

reduction. Pathway analysis revealed the contribution of these functional gene groups

13

along a depth gradient. In addition, denitrification processes increased, while anammox

14

processes decreased consistent with a rise in temperature from 4 to 12 °C. Furthermore,

15

cold temperatures exhibited different effects on anammox and denitrification and their

16

long-term acclimatization capacities changed with temperature.

17 18

3



19

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INTRODUCTION

20

Nitrogen is one of the primary pollutants in wastewater and gives rise to

21

eutrophication in aquatic environments and subsequent toxicity to aquatic organisms.1, 2

22

During the last few decades, constructed wetlands (CWs) have been applied to nitrogen

23

(from wastewater) removal due to the unique advantage of low-cost and low-energy

24

consumption,3-5 whereas CWs are often unsatisfactory in nitrogen removal rates.4

25

Recently, CWs with 'tidal flow' were developed, which improved nitrogen removal

26

performance.5, 6

27

In tidal flow CWs (TFCWs), a rhythmic cycle of flood/drain is generated, providing

28

better oxic-anoxic conditions for nitrification and denitrification processes.7,

8

29

Ammonium cations (NH4+-N) are first adsorbed on negatively charged surfaces in the

30

flood cycle. Air is immediately brought into the system as it drains, stimulating the

31

adsorbed NH4+-N nitrified. Finally, in the next flood cycle, nitrate (NO3--N) and nitrite

32

(NO2--N) desorb into bulk water, where denitrification takes place and NO3--N and NO2-

33

-N convert to atmospheric nitrogen.9 Nevertheless, some studies reported limited

34

denitrification processes in TFCWs, resulting in NO3--N accumulation, while NH4+-N

35

removal rates were high.9-11 The aerobic environment in TFCWs might be

36

disadvantageous to denitrifiers and the carbon sources necessary for denitrification are

37

insufficient, as the organic matter is preferentially degraded, leading to NO3--N

38

accumulation.11 As a result, denitrification can be regarded as a rate-limiting total

39

nitrogen (TN) removal process in TFCWs.

40

In addition to nitrification and denitrification, anammox can be another pathway to

41

nitrogen removal in TFCWs. Hu et al. built a single stage TFCW using internal

42

recirculation, which achieved total nitrogen removal of > 80% via the complete

43

autotrophic nitrogen removal over nitrite (CANON) process.12 Moreover, previous 4


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44

studies demonstrated the presence of simultaneous nitrification, anammox, and

45

denitrification (SNAD) processes in a TFCW through molecular analyses and revealed

46

C/N ratios and flood/drain time ratios could affect TFCW nitrogen removal pathways.13-

47

15

48

To date, most TFCW studies were conducted at room temperature and only a few

49

studies examined treatment performance under cold temperature regimes.16 Cold

50

temperatures have always been problematic in CWs; impacting microbial metabolism

51

and growth17 with subsequent depressed nitrogen removal.18 Little is known about

52

nitrogen transformation pathways in CWs under cold temperatures, therefore TFCWs

53

require even more research to elucidate cold temperature denitrification processes.

54

Denitrification is commonly considered inhibited below 15 °C and below 5 °C, the

55

process terminates.19, 20 However, some studies reported denitrification at 4 °C or even

56

lower, with lower activity rates observed.21 In addition, cold temperatures below 15 °C

57

could harm anammox,22 and anammox processes were negligible in reactions below 10

58

°C.23 Nevertheless, TFCWs and nitrogen transformation processes must be examined to

59

address the interactions, inhibitions, and capacity to acclimate under cold temperature

60

conditions.

61

The present study serves to fill this knowledge gap and is the first to explore the

62

underlying molecular mechanisms of nitrogen transformation in TFCWs under cold

63

temperatures. We explored the following objectives to elucidate nitrogen transformation

64

in TFCWs: 1) evaluate the long-term effects of three cold temperature regimes (4, 8, and

65

12 °C) on nitrogen transformation rates; 2) determine the key functional gene groups

66

underlying denitrification processes under the three cold temperatures; 3) assess the

67

contribution of key functional gene groups along a depth gradient. The results of this

68

study might have significant implications for nitrogen removal enhancement in TFCWs 5



69

under cold temperatures.

70

MATERIALS AND METHODS

71

Construction and operation of TFCWs. Three TFCWs (termed T1, T2, and T3)

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with an effective working volume of 40 L (L × W × H = 120 × 40 × 20 cm) were

73

constructed and filled with 8-10 mm diameter lava rock. Canna indica L. (Cannaceae)

74

as an herbaceous species with ornamental value, widely used in CWs in northern China,

75

24

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cooling pipes with a total length of 15 m and inside diameter of 5 mm were coiled inside

77

the TFCWs and connected to an external cooling chiller. TFCWs were covered with

78

insulation boards and cotton quilts to ensure adequate insulation. Two PVC pipes (L × D

79

= 100 × 5 cm2) were pre-buried in each TFCW for microbial sampling and molecular

80

analyses following Zhi and Ji.14

was planted on the surface of each TFCW at an initial density of 22 plants/m2. Copper

81

T1, T2, and T3 were fed with synthetic wastewater, which contained 50 mg/L

82

chemical oxygen demand (COD), ~8mg/L NH4+-N, and ~5mg/L NO3--N. KH2PO4,

83

FeCl3·7H2O, CaCl2, NaHCO3, and MgSO4·7H2O were added at 4.0, 3.0, 2.0, 23, 12, and

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100 mg/L concentrations, respectively. The water pH was between 7.0 and 7.4.

85

During start-up stage of T1, T2 and T3, we inoculated 100 g high efficiency

86

compound microbial inoculum B350M (Bio-systems Co.) into each TFCW.25 T1, T2,

87

and T3 were operated under 4, 8, and 12 °C, respectively. The flood-and-drain cycle was

88

repeated every 36 h, providing 24 h of wastewater-bed contact in a "wet" phase and 12 h

89

of re-oxygenation in a "dry" phase, based on Zhi and Ji.14 The experiment was initiated

90

on 5 October 2012 and involved the following stages: 14 d of start-up, 55 d of

91

domestication, and 127 d of long-term operation.

92

Sample collection and determination. During the operation phase, water samples

93

were collected from T1, T2, and T3 under a depth gradient of 0-20 cm, 20-40 cm, 40-60 6


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cm, 60-80 cm, and 80-100 cm every two weeks, and analyzed immediately at the Key

95

Laboratory of Water and Sediment Sciences at Peking University. COD, NH4+-N, NO2--

96

N and NO3--N determination were conducted according to standard analytical

97

procedures.26 COD was measured using a HACH DR2800 (HACH, Loveland, Colorado,

98

USA).

99

spectrophotometer (Shimadzu, Kyoto, Japan).

NH4+-N,

NO2--N,

and

NO3--N

were

determined

with

a

UV-1800

100

Microbial samples were collected biweekly from each T1, T2, and T3 layer of the

101

TFCW from days 70 to 196. During each sampling event, the pre-buried columns were

102

removed from each TFCW bed, placed in an ice incubator, and immediately sent to the

103

laboratory for DNA extraction. Soil DNA kits D5625-01 (Omega, USA) were used to

104

extract and purify total genomic DNA from the samples. Extracted genomic DNA was

105

detected by 1% agarose gel electrophoresis and stored at -20 °C.

106

Detection of nitrogen functional genes. An abundance of bacterial 16S rRNA,

107

anammox bacteria 16S rRNA; nitrogen functional genes, including ammonia

108

monooxygenase (amoA), nitrite oxidoreductase (nxrA), periplasmic nitrate reductase

109

(napA) and membrane-bound nitrate reductase (narG), nitrite reductase (nirS), nitric

110

oxide reductase (qnorB), and nitrous oxide reductase (nosZ) were identified.

111

Quantitative polymerase chain reaction (qPCR) using a MyiQ2 real-time PCR Detection

112

System (Bio-Rad, USA) with the fluorescent dye SYBR-Green approach was employed

113

in rRNA and functional gene amplification. The amplification of qPCR was performed

114

in 20 µL reaction mixtures, including 10 µL of SYBR Green I PCR master mix (Applied

115

Biosystems, USA), 1µL of template DNA (sample DNA or plasmid DNA for standard

116

curves), 0.5µL of forward and reverse primers, and 8µL of sterile water. Each qPCR

117

amplification comprised 40 total cycles. The primer sequences and qPCR protocol

118

followed Ji et al.27 7



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Statistical Analyses. Influent and effluent concentrations and hydraulic retention

120

time (HRT) (36 h) were used to calculate NH4+-N, NO3--N, NO2--N, and TN removal

121

efficiencies and transformation or accumulation rates. TFCW nitrogen transformation

122

rates at each sampling time (biweekly) were averaged over the five depths. Relative

123

abundances were determined as the abundances of functional genes divided by the

124

abundance of bacterial 16S rRNA. Standard deviations (S.D.) of gene relative

125

abundance data were calculated using three replicates measured in qPCR and plotted in

126

Fig. S1 as error bars for assessing data variations and measurement errors. The

127

quantitative response relationships between nitrogen transformation rates and functional

128

gene groups were determined via stepwise regression models using SPSS Statistics 20

129

(IBM, USA). An a priori P-value of P < 0.05 was defined to establish significant

130

effects. Contribution of different functional gene groups to nitrogen transformation rates

131

was derived from path analysis, as described in Wang et al.28 Direct effects (path

132

coefficients) were obtained by the simultaneous solution of the normal equations for

133

multiple linear regression in standard measure.29 Indirect effects were derived from

134

simple correlation coefficients between functional genes using SPSS Statistics 20 (IBM,

135

USA).

136

RESULTS AND DISCUSSION

137

Nitrogen removal and transformation. During the operation phase (70-196 d),

138

T1, T2, and T3 achieved high and stable NH4+-N removal efficiencies (93 - 96%) , with

139

NH4+-N removal loading rate ranging from 10.2 to 10.5 g/m3/d (Fig. 1). Lee et al.

140

reported only 45% NH4+-N removal efficiency and 2.8 g/m3/d NH4+-N removal rate in a

141

typical vertical subsurface flow CW under 5 °C, which was 20% lower removal

142

efficiency than under 23 °C.30 In a two-stage TFCW with a 24 h "wet" and 12 h "dry"

143

phases, NH4+-N removal rate achieved 7.70 g/m3/d when temperatures were ～5-10 °C.31 8


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Zou et al. built a single-stage TFCW with a 21 h hydraulic retention time (HRT), of

145

which NH4+-N removal rate was ～10.0 g/m3/d, at a mean 5 °C.32 Compared to these

146

other CW studies, T1, T2, and T3 achieved equivalent or even higher NH4+-N removal

147

efficiencies and rates. In addition, NH4+-N transformation rates exhibited little variation

148

(P 0.05) in the rates between different depth

178

gradient. In T3, the NO3--N accumulation rate in the 0-20 cm layer was significantly

179

lower than that in other layers (P = 0.011 < 0.05), while the rates varied little (P > 0.05)

180

along the depth gradient from 20 to 100 cm.

181

Based on these results, we concluded NO3--N accumulation was the major factor

182

affecting TN removal. As NO3--N accumulation rates decreased consistent with a rise in

183

temperature from 4 to 12 °C, TN transformation rates and removal efficiencies increased

184

(Fig. 1). Furthermore, TN removal was enhanced by long-term TFCW operation

185

facilitating NO3--N reduction recovery (Fig. 2).

186

Quantitative response relationships. In this study, functional gene group ratios

187

(ratio or summation of different functional genes) were used as variables and introduced

188

into stepwise regression analyses. Results showed nitrogen transformation in the period

189

before 126 d differed from after 126 d, (see the previous section); therefore we

190

investigated the functional gene groups in 70-126 d and 127-196 d periods separately to

191

better characterize the nitrogen transformation pathway during different time periods.

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nirS/amoA was the key factor for NH4+-N transformation in T2 and T3 and showed a

193

positive relationship with NH4+-N transformation rates (Table 1). The amoA and nirS 10


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194

genes are often regarded as NH4+-N to NO2--N oxidation and NO2--N to NO reduction

195

markers, respectively.34, 35 Also, amoA and nirS were respectively involved in the two

196

consecutive steps of nitrification and denitrification. Thus, nirS/amoA can be denoted as

197

NO2--N level consumption by denitrification. The more NO2--N consumed, the more

198

NH4+-N transformed, because lower NO2--N concentrations can reduce its toxic effects

199

on ammonia-oxidizing bacteria (AOB).36, 37 Results showed nitrification coupled with

200

denitrification was the primary NH4+-N removal pathway under 8 and 12 °C. nirS/amoA

201

was the rate-limiting factor during 70-126 d in T1, however rates were limited by

202

anammox/amoA after 126 d (Table 1). anammox/amoA was a coupling factor between

203

partial nitrification and anammox, which was named CANON (Completely Autotrophic

204

Nitrogen removal Over Nitrite).38 CANON exhibited a positive relationship with NH4+-

205

N transformation rates during 127-196 d, indicating the main NH4+-N removal pathway

206

changed from nitrification coupling with denitrification to CANON under 4 °C.

207

amoA/(narG+napA) was the key factor responsible for NO2--N accumulation in T1

208

during the entire operation phase (Table 1). narG and napA were regarded as marker

209

genes for NO3--N into NO2--N reduction and responsible for the first denitrification step.

210

Generally, narG is dominant under anoxic and napA under oxic conditions.39, 40 Hence,

211

amoA/(narG+napA) denoted NO2--N accumulation, as amoA and narG/napA genes

212

were both primarily involved in NO2--N production, showed a positive relationship with

213

NO2--N accumulation rates. This result suggested nitrification and denitrification

214

processes affected NO2--N accumulation at 4 °C. The T2 rate-limiting factor during 70-

215

126 d was the same in T1 (Table 1). After 126 d, qnorB/(narG+napA) became the key

216

factor and showed a negative relationship with NO2--N accumulation rates. The qnorB

217

gene was regarded as the NO into N2O conversion marker and involved in the third

218

denitrification step.41 These indicated denitrification processes alone determined NO2--N 11



219

accumulation during 127-196 d at 8 °C. napA/bacteria was the major factor in T3 during

220

70-126 d and showed a positive relationship with NO2--N accumulation rates, while

221

qnorB/napA was the primary factor during 127-196 d, and exhibited a negative

222

relationship with NO2--N accumulation rates (Table 1). These results indicated napA

223

played a more important role in NO2--N accumulation at 12 °C compared with 4 and 8

224

°C. napA abundance in T3 showed a 1.9-fold increase relative to T2 and a 3.0-fold

225

increase relative to T1 (Fig. S1), suggesting lower temperatures were not conducive to

226

napA. In addition, narG showed increased abundance compared with napA in T3 (Fig.

227

S1), however results suggested napA instead of narG determined NO2--N accumulation.

228

Moreover, napA is often regarded as the functional gene involved in dissimilatory nitrate

229

reduction to ammonia (DNRA),42 indicating that in addition to denitrification, DNRA

230

might be another pathway contributing to NO2--N accumulation at 12 °C, because of the

231

increased importance of DNRA under warmer condition (compared to 4 and 8 °C).43

232

Nevertheless, due to the relative low NH4+-N (0.3-0.5mg/L) and NO2--N (0.2-0.6mg/L)

233

effluent concentration in the TFCWs, as well as the low C/N ratios (a C/N ratio > 6 was

234

necessary for DNRA15), DNRA was not significant in our TFCWs.

235

Functional gene group determination in relationship to NO3--N accumulation varied

236

under different temperature regimes (Table 1). nxrA/amoA was the determining factor

237

from 70 to 126 d in T1, showing a positive relationship with NO3--N accumulation rates.

238

nxrA was regarded as the NO2--N to NO3--N oxidation marker involved in the second

239

nitrification step.44 Therefore, nxrA/amoA was denoted as the completed nitrification

240

process, indicating NO3--N accumulation rates were determined by nitrification before

241

126 d. CANON (i.e., anammox/amoA) was the key factor following 126 d in T1, and

242

exhibited a negative relationship with NO3--N accumulation rates. As the CANON ratio

243

gradually increased from 26.4 to 37.7 (Fig. S2), NO3--N accumulation rates decreased 12


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244

from 7.62 to 1.76 g/m3/d (Fig. 2a). Compared with traditional nitrification processes,

245

CANON produced less NO3--N.45 However, a NO3--N (10%) quantity was still released

246

from CANON.46 As a result, NO3--N remained accumulated in T1 when the operation

247

phase was terminated. In conclusion, as the operation phase proceeded, the major

248

pathways affecting NO3--N accumulation under 4 °C changed from nitrification to

249

CANON.

250

nxrA/(narG+napA) was responsible for NO3--N accumulation in T2 during the 70-126

251

d time period and showed a positive relationship with NO3--N accumulation rates.

252

(narG+napA)/amoA replaced nxrA/(narG+napA) during 126-196 d and showed a

253

negative relationship with NO3--N accumulation rates. We concluded that nitrification

254

and denitrification were the primary pathways affecting NO3--N accumulation under 8

255

°C during the entire operation phase.

256

(narG+napA)/bacteria and nirS/bacteria were the key factors controlling accumulation

257

rates in T3 during the 70-126 d period, while (narG+napA)/bacteria alone was

258

responsible after 126 d. (narG+napA)/bacteria exhibited a negative relationship with

259

NO3--N accumulation rates in 70-126 d and 127-196 d. As the (narG+napA)/bacteria

260

ratio increased from 1.38×10-5 to 7.94×10-5 (Fig. S2), the NO3--N accumulation rates

261

decreased from 3.60 g/m3/d to -0.63 g/m3/d (Fig. 2c). Results indicated denitrification

262

was the dominant pathway under 12 °C. Furthermore, NO3--N accumulation was

263

influenced by total bacterial 16S rRNA abundance at 12 °C, suggesting the TFCW

264

bacterial community contributing to NO3--N accumulation increased in complexity at 12

265

°C. This was shown by a rise in temperature, enhanced bacterial species richness47 and

266

competition between denitrifiers and other heterotrophic microbes.48

267

Nitrogen transformation pathway. Path analysis was employed to assess the

268

contribution of functional gene groups to nitrogen removal in T1, T2, and T3. The 13



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269

positive or negative value of the effects indicated the functional gene groups contributed

270

to NO3--N accumulation or reduction.

271

A higher direct effect of nxrA/amoA (0.82) on NO3--N accumulation rates compared

272

with anammox/amoA (-0.20) was observed in T1 from 70 to 126 d (Fig. 4a). After 126 d,

273

anammox/amoA played a dominant role, and the direct effect increased to -0.98. These

274

results suggested the nxrA gene might compete with anammox bacteria for the NO2--N

275

substrate produced by the amoA gene during 70-126 d. Generally, nitrite-oxidizing

276

bacteria (NOB), which contain the nxrA gene, grow at temperatures ranging from -2 to

277

15 °C.49, 50 However, anammox process was found inhibited when temperatures were

278

lower than 15 °C.22 High nxrA activity was detrimental to nitrogen removal because

279

nxrA takes up limited O2 and NO2--N before aerobic and anaerobic ammonium-

280

oxidizing bacteria, facilitating NO3--N accumulation.51 nxrA relative abundance

281

decreased by 44% while anammox doubled in T1 during 127-196 d compared with 70-

282

126 d (Fig. S1), indicating the anammox bacteria slowly acclimated to the cold

283

environment and subsequently out-competed the nxrA gene by quantity. Previous studies

284

reported anammox acclimation required a long period of time,51,

285

adaptation of anammox bacteria appeared after a long operation phase under cold

286

temperatures (below 10 °C) in a subsurface flow CW,22 which were congruent with the

287

present study.

52

and obvious

288

The direct effects of nxrA/(narG+napA) on NO3--N accumulation rates in T2

289

decreased from 0.65 (70-126 d) to 0.08 (126-196 d) (Fig. 4b). In contrast, the direct

290

effects of (narG+napA)/amoA increased from -0.37 to -0.96 (Fig. 4b). The direct effects

291

of (narG+napA)/bacteria on NO3--N accumulation rates in T3 increased from -0.86 to -

292

0.94 (Fig.4c). The first step in denitrification was a primary pathway responsible for

293

NO3--N accumulation in T2 and T3 during the entire operation phase (70 to 196 d). 14


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294

The

above

results

indicated

anammox/amoA,

(narG+napA)/amoA,

and

295

(narG+napA)/bacteria were the key factors contributing to enhancement of NO3--N

296

reduction in T1, T2, and T3, respectively. The contributions of these functional gene

297

groups under the different temperatures are shown in Figure 4 (a), (b), and (c). Although

298

CANON was the dominant factor at 4 °C (-0.899), (narG+napA)/bacteria had a direct

299

effect (-0.165) on NO3--N accumulation, suggesting denitrification processes remained a

300

minor pathway at 4 °C. CANON effects decreased to -0.397 at 8 °C and -0.04 at 12 °C,

301

whereas (narG+napA)/bacteria increased to -0.93 at 12 °C. Therefore, the contribution

302

of denitrification was increasing, while CANON processes were decreasing with the rise

303

in temperature from 4 to 12 °C. Similar results were found in previous studies that

304

compared to denitrification, anammox exhibited better cold adaptation,53-55 and

305

anammox was more specialized for psychrophilic.54

306

Path analysis was applied to examine how the dominant functional gene groups

307

affected nitrogen removal in different layers of the TFCWs. Figure 3 (a), (b), and (c)

308

shows the effects of anammox/amoA, (narG+napA)/amoA, and (narG+napA)/bacteria

309

on NO3--N accumulation rates along the depth gradient in T1, T2, and T3, respectively.

310

CANON exhibited the greatest effects on NO3--N accumulation rates (-0.82) in T1 for

311

the 20-40 cm layer and sharply fell to -0.37 in the 40-60 cm layer. It slowly increased to

312

-0.62 in the 60 to 100 cm depth. Similarly, T1 exhibited low NO3--N accumulation rates

313

in the 20-40 cm layer and higher rates in the 40-60 cm layer (Fig. 3). These results

314

demonstrated CANON contribution was highest in the 20-40 cm layer. CANON

315

processes can be affected by dissolved oxygen (DO) and lower DO levels facilitated

316

CANON.56 In the TFCWs, DO gradually decreased consistent with depth.9 However

317

from 0-60 cm, CANON contributions did not exhibit the expected changes based on DO

318

levels, therefore some other factors (NH4+-N concentration or organic matter57, 58) might 15



319

drive CANON processes and further investigations are required to identify the

320

mechanisms.

321

The functional gene group contributions decreased from 0 to 60 cm in depth for T2

322

and T3 and the highest values were detected in the 0-20 cm layer (Fig. 3). The 0-20 cm

323

layer was considered the rhizosphere,59 where dead roots shed material and organic

324

matter is exuded by roots, providing a significant source of nutrients for heterotrophic

325

bacteria.60 Consequently, this layer was beneficial to denitrifiers and NO3--N showed

326

decreased accumulation levels. The depth change to the 60 to 100 cm layer resulted in

327

increased (narG+napA)/amoA and (narG+napA)/bacteria effects in T2 and T3,

328

respectively.

329

Denitrification and anammox inhibition and acclimatization. Previous studies

330

investigated CW denitrification inhibition under cold temperature regimes. Poe et al.

331

demonstrated a notable denitrification rate decreases in CWs when the seasonal

332

temperature remained less than 15 °C.61 Hajaya et al. reported nitrate reduction rates

333

were severely affected by 10 °C and below in a surface flow constructed wetland.62

334

These studies only investigated denitrification performance in CWs under cold

335

temperatures, however the molecular mechanisms of inhibited denitrification were not

336

explored. Furthermore, Vacková et al. showed microorganisms involved in

337

denitrification acclimated to cold temperatures;63 nevertheless, few studies have

338

examined the acclimatization of denitrification processes in CWs.

339

In this study, we found a denitrification inhibition and acclimatization process under

340

4, 8 and 12 °C. The first denitrification step (reduction of nitrate to nitrite) was a rate-

341

limiting pathway of nitrate reduction in T2 and T3 (Fig. 3). Therefore, as NO3--N

342

accumulation rates were initially high and subsequently reduced to almost zero near the

343

end of the operation phase, denitrification was first inhibited by cold temperatures and 16


Page 16 of 34

Page 17 of 34


344

then gradually acclimated. The mean values of (narG+napA)/bacteria during 127-196 d

345

increased 2.1-fold (T1), 3.1-fold (T2), and 3.6-fold (T3) relative to the first 126 d (Fig.

346

S1), suggesting lower temperatures had negative effects on the first step in

347

denitrification, including acclimatization and overall susceptibility to cold temperatures

348

compared with other denitrification steps. Similarly, Misiti et al. reported reduction rates

349

exceeding 60% in nitrate and nitrite as temperatures decreased from 10 to 5 °C, whereas

350

nitric and nitrous oxide reduction rates exhibited little change.64 A primary reason for

351

denitrifier inhibition under lower temperatures is decreased affinity for substrates,65

352

which have an even more serious impact on denitrification when insufficient

353

biodegradable carbon conditions exist.64 Furthermore, Pan et al. showed nitrate

354

reductase exhibited a lower capacity to compete for electrons compared to nitrite

355

reductase.66 Therefore, as the C/N ratio was insufficient in our TFCWs, lower

356

temperatures might damage the first denitrification step.

357

Previous reports indicated anammox activity completely ceased at temperatures below

358

10 °C and anammox processes were negligible in reactors;23 however several studies

359

conducted

360

temperatures.67 Risgaard-Petersen et al. examined sediments from Greenland's east and

361

west coasts and observed anammox activity between −2 and 30 °C, with optimum

362

temperature at 12 °C.68 Dalsgaard and Thamdrup evaluated marine sediments from the

363

Skagerrak (Baltic-North Sea) and reported anammox was responsible for approximately

364

80% of the total nitrogen gas production at 6 °C.69 In the present study, the mean relative

365

abundance of anammox bacteria during 70-126 d at 4 °C was nearly half that at 8 and 12

366

°C, and the mean relative abundance of anammox bacteria doubled after 126 d (Fig. S1).

367

These results suggested anammox was more affected by 4 °C than 8 and 12 °C in the

368

short term (70-126 d). Inhibition of anammox and denitrification processes at 4 °C

with

marine

anammox

reported

measurable

17


activity

under

cold


Page 18 of 34

369

might be responsible for the high NO3--N accumulation rates in T1 compared with rates

370

in T2 and T3 at the beginning of the operation phase. However, long-term acclimation at

371

4 °C might improved the cold resistance of anammox bacteria.22 CANON instead of

372

denitrification processes played a dominant role in T1 after 126 d (Fig. 3). We

373

speculated anammox might have a greater capacity to acclimate under 4 °C conditions,

374

compared with denitrification. In contrast, following the strengthening of denitrification

375

processes at 8 and 12 °C, the role of CANON was decreased.

376

Previous studies revealed simultaneous nitrification, anammox, and denitrification

377

(SNAD) processes were major nitrogen removal pathways in TFCWs13,

378

temperature. In the present study, cold temperature inhibited anammox and

379

denitrification processes at variable levels, and acclimatization under long-term

380

operation changed with temperature. Further study should be performed to accurately

381

evaluate anammox and denitrification rates using stable isotope techniques (15N).

382

Overall, our study generated insights into the mechanisms of enhanced long-term

383

nitrogen removal, which might provide significant contributions to improve nitrogen

384

removal processes under cold temperature conditions.

385 386 387 388 389 390 391 392 393

18


15

at room

Page 19 of 34


394

395

The National Natural Science Foundation of China (No. 51179001), and the

396

Collaborative Innovation Center for Regional Environmental Quality provided support

397

for this study.

Acknowledgements

398 399

400

(1) Camargo, J. A., Ecological and toxicological effects of inorganic nitrogen pollution

401

in aquatic ecosystems: A global assessment. Environ. Int. 2006, 32, (6), 831–849.

402

(2) Rabalais, N. N., Nitrogen In Aquatic Ecosystems. Ambio. 2002, 31, (2), 102-112.

403

(3) Saeed, T.; Sun, G. Z., A review on nitrogen and organics removal mechanisms in

404

subsurface flow constructed wetlands: Dependency on environmental parameters,

405

operating conditions and supporting media. J. Environ. Manage. 2012, 112, 429-448.

406

(4) Lee, C. G.; Fletcher, T. D.; Sun, G. Z., Nitrogen removal in constructed wetland

407

systems. Eng. Life Sci. 2009, 9, (1), 11-22.

408

(5) Sun, G. Z.; Zhao, Y. Q.; Allen, S., Enhanced removal of organic matter and

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ammoniacal-nitrogen in a column experiment of tidal flow constructed wetland system.

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J. Biotechnol. 2005, 115, (2), 189-197.

411

(6) Vymazal, J., Constructed wetlands for wastewater treatment: five decades of

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experience. Environ. Sci. Technol. 2011, 45, (1), 61-69.

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(7) Cui, L. H.; Feng, J. K.; Ouyang, Y.; Deng, P. W., Removal of nutrients from septic

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effluent with re-circulated hybrid tidal flow constructed wetland. Ecol. Eng. 2012, 46,

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(8) Wu, S. B.; Zhang, D. X.; Austin, D.; Dong, R. J.; Pang, C. L., Evaluation of a lab-

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matter and ammonium removal. Ecol. Eng. 2011, 37, (11), 1789-1795.

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(9) Chang, Y. J.; Wu, S. B.; Zhang, T.; Mazur, R.; Pang, C. L.; Dong, R. J., Dynamics of

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nitrogen transformation depending on different operational strategies in laboratory-scale

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(68) Risgaard-Petersen, N.; Meyer, R. L.; Schmid, M. C.; Jetten, M. S. M.; Enrichprast,

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600 601

25



602

Captions

603

Table 1. Quantitative response relationships between nitrogen transformation rates and

604

functional gene groups in T1, T2, and T3.

605

Fig. 1. Temperature effects on NH4+-N and TN removal efficiencies (column) and NH4+-

606

N, NO3--N, and NO2--N mean transformation rates (symbol and line).

607

Fig. 2. Daily changes in NH4+-N, NO3--N, NO2--N, and TN transformation rates at 4 °C

608

(a), 8 °C (b), and 12 °C (c).

609

Fig. 3. Variation in the contribution of functional gene groups and averaged NO3--N

610

transformation rates along a depth gradient. CANON (anammox/amoA) at 4 oC (a),

611

(narG+napA)/amoA at 8 oC (b), (narG+napA)/16S rRNA at 12 oC (c), triangle, circle,

612

and rectangle (d) denote 4 oC, 8 oC, and 12 oC, respectively.

613

Fig. 4. Path diagrams showing functional gene group effects on NO3--N accumulation

614

rates under different temperature regimes.

615

26


Page 26 of 34

Page 27 of 34


Table 1. Quantitative response relationships between nitrogen transformation rates and

functional gene groups in T1, T2 and T3 R2

Stepwise regression equations

P

(70-126 d, n=5) T1

Stepwise regression equations

R2

P

0.847

0.009

0.953

0.001

0.819

0.013

0.918

0.003

0.821

0.013

0.722

0.032

0.883

0.005

0.875

0.006

0.864

0.007

(127-196 d, n=6)

NH4+-N =0.044

+ 9.856

0.798

NO3--N = 1.395 + 7.698

0.976

0.041

NH4+-N =0.110

+ 10.017

(4 °C)

0.002

NO3--N = -0.504

+

20.440

-

NO2 -N = 1.082 + 0.132 T2

NH4+-N =0.385 + 9.198

0.939

0.007

NO2--N = 0.981 + 0.141

0.786

0.045

NH4+-N =0.066 + 9.825

0.879

0.019

NO3--N = -0.029

(8 °C)

NO3--N = 38.408

+ 4.906

NO2--N = 0.499 + 0.471

0.831

0.031

+ 5.364

NO2--N = -0.480 + 0.623

T3

NH4+-N = 0.132 + 9.940

0.824

0.033

NH4+-N =0.055 + 10.127

0.973

0.027

NO3--N = -6.217×104

(12 °C) NO3--N = -5.702×104 2.607×104

-

+

5.207

+ 4.866

NO2--N = 4.204×105 + 0.165

0.928

0.009

NO2--N = - 0.180

27


+ 0.585


Fig. 1. Temperature effects on NH4+-N and TN removal efficiencies (column) and NH4+-

N, NO3--N, and NO2--N mean transformation rates (symbol and line).

28


Page 28 of 34

Page 29 of 34


Fig. 2. Daily changes in NH4+-N, NO3--N, NO2--N, and TN transformation rates at 4 °C

(a), 8 °C (b), and 12 °C (c).

29



Fig. 3. Variation in the contribution of functional gene groups and averaged NO3--N

transformation rates along a depth gradient. CANON (anammox/amoA) at 4 oC (a), (narG+napA)/amoA at 8 oC (b), (narG+napA)/16S rRNA at 12 oC (c), triangle, circle,

and rectangle (d) denote 4 oC, 8 oC, and 12 oC, respectively.

30


Page 30 of 34

Page 31 of 34


Fig.4. Path diagrams assessing the effects of functional gene groups on NO3--N

accumulation rate under different temperatures.

31



Supporting Information

Fig. S1 Relative abundance of functional genes during different periods at the

temperature of 4 °C, 8 °C and 12 °C. Fig. S2 Dynamic ratio of functional gene groups at the temperature of 4 °C (a), 8 °C (b)

and 12 °C (c).

32


Page 32 of 34

Page 33 of 34


Fig. S1 Relative abundance of functional genes during different periods at the

temperature of 4 °C, 8 °C and 12 °C

33



Fig. S2 Dynamic ratio of functional gene groups at the temperature of 4 °C (a), 8 °C (b)

and 12 °C (c).

34


Page 34 of 34

Cold Temperature Effects on Long-Term Nitrogen Transformation

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