A) for enhanced nitrogen removal from mature landfill leachate

Journal Pre-proofs A continuous-flow combined process based on partial nitrification-Anammox and partial denitrification-Anammox (PN/A+PD/A) for enhanced nitrogen removal from mature landfill leachate Zhong Wang, Liang Zhang, Fangzhai Zhang, Hao Jiang, Shang Ren, Wei Wang, Yongzhen Peng PII: DOI: Reference:

S0960-8524(19)31713-4 https://doi.org/10.1016/j.biortech.2019.122483 BITE 122483

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Bioresource Technology

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9 October 2019 20 November 2019 21 November 2019

Please cite this article as: Wang, Z., Zhang, L., Zhang, F., Jiang, H., Ren, S., Wang, W., Peng, Y., A continuousflow combined process based on partial nitrification-Anammox and partial denitrification-Anammox (PN/A+PD/ A) for enhanced nitrogen removal from mature landfill leachate, Bioresource Technology (2019), doi: https:// doi.org/10.1016/j.biortech.2019.122483

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1

A continuous-flow combined process based on partial

2

nitrification-Anammox and partial denitrification-Anammox

3

(PN/A+PD/A) for enhanced nitrogen removal from mature landfill

4

leachate

5

Zhong Wang a, Liang Zhang b, Fangzhai Zhang b, Hao Jiang b, Shang Ren b, Wei Wang

6

c,

7

a. State Key Laboratory of Urban Water Resource and Environment, Harbin Institute

8

of Technology, Harbin 150090, China

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b. National Engineering Laboratory for Advanced Municipal Wastewater Treatment

Yongzhen Peng a, b,*

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and Reuse Technology, Beijing University of Technology, Beijing 100124, China

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c. College of Civil and Architectural Engineering, Heilongjiang Institute of

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Technology, Harbin 150050, China

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* Corresponding author：Yongzhen Peng

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E-mail address: [email protected]

1

15 16

Abstract: A novel continuous-flow combined process of partial nitrification, Anammox

17

(PN/A) and partial denitrification-Anammox(PD/A) was established to achieve

18

enhanced nitrogen removal from landfill leachate. The NH4+-N transformation rate

19

and NO2--N accumulation rate in the PN reactor reached 93.4% and 91.5%,

20

respectively. The nitrite generated from the PN reactor was combined with influent

21

(38%) and fed into the Anammox reactor. The nitrate produced in the Anammox

22

reactor was then discharged to PD/A reactor, where nitrate was transformed to nitrite

23

and removed via Anammox. Under a COD/NO3--N ratio of 4.0, the NO3--N

24

-to-NO2--N transformation ratio (NTR) and Anammox contribution rate reached 60.4%

25

and 57.1% in PD/A reactor. The final effluent TN concentration was 15.7mg/L, and

26

the efficiency of TN removal could reach 98.8%. By combining PN/A with PD/A,

27

enhanced nitrogen removal from landfill leachate was achieved successfully with an

28

external carbon source addition (COD/NH4+-N) of 0.28.

29

Keywords:

30

Landfill leachate; Partial nitrification; Anammox; Partial denitrification; Enhanced

31

nitrogen removal

32

1 Introduction

33

Landfill disposal is a common method of municipal solid waste management

34

worldwide, as it is low cost and requires relatively low maintenance (Iskander et al.,

35

2018). Leachate is produced in the process of the solid waste stabilization and water

36

infiltration, requiring appropriate treatment before discharge. Due to the high

2

37

concentrations of ammonia (>1000mg/L) and organic/inorganic matter

38

(2200-13500mg/L) in leachate, cost-effective treatment methods for landfill leachate

39

remain a global challenge in wastewater engineering (Zhou et al., 2018). Therefore,

40

investigating economical and effective methods for the treatment of leachate is a

41

continuing concern within the field of wastewater treatment.

42

In conventional biological nutrient removal processes such as A2/O process, high

43

concentrations of ammonia can be removed from leachate through nitrification and

44

denitrification processes. However, a large amount of external carbon is required for

45

denitrification, resulting in excessive sludge production. Anaerobic ammonium

46

oxidation (Anammox) has the unique capacity to remove ammonium and nitrite to

47

nitrogen gas, and has excellent potential as an alternative to conventional treatment

48

methods(Kuenen, 2008). Since the discovery of Anammox in the 1990s, the

49

Anammox process has attracted much research interest for its high-efficiency and

50

low-cost nitrogen removal capability(Mulder et al., 1995). Recently, a growing body

51

of literature has recognized the potential value of the Anammox process in the

52

treatment of leachate(Miao et al., 2019; Wang et al., 2016b). The removal efficiency

53

of COD and nitrogen from leachate can simultaneously reach 68.5% and 82.4%,

54

respectively, under SNAD processes(Wang et al., 2018). Using a combined

55

Sharon-Anammox process treating mature leachate, 84% total nitrogen removal, and

56

71% ammonia nitrogen removal was achieved in 147 days(Sri Shalini & Joseph,

57

2018).

58

However, nitrate is inevitably produced during the nitrification and Anammox

3

59

process. Theoretically, 11% of TN may remain in the form of NO3--N during the

60

PN/A process, presenting a significant limitation as high amounts of NO3--N in the

61

effluent (>100mg/L) would not meet discharge standard requirements (TN<20mg/L).

62

Due to the low BOD5/COD and high concentration of ammonia, large amounts of

63

external carbon source are required to remove the remaining NO3--N during complete

64

denitrification, increasing the cost of treatment and resulting in excessive sludge

65

production. In previous studies, partial denitrification (PD) is an economical method

66

to deal with NO3--N -rich wastewater (Kalyuzhnyi et al., 2006; Kartal et al., 2007;

67

Wang et al., 2019). Compared with nitrification/denitrification process, the PD/A

68

process may be a promising nitrogen removal process for its low organic matter

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demand (79% reduced) and low oxygen demand (45% reduced)(Ma et al., 2016).

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Recently, the integration of PD and Anammox has been presented as a potential

71

alternative method for the treatment of wastewater containing ammonium and nitrate

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in single and two-stage reactors, with a high TN removal efficiency (Du et al., 2019;

73

Li et al., 2017). Moreover, in an upgraded municipal WWTP consisting of anoxic

74

MBBR and AAO system, the enhanced nitrogen removal through PD/A and

75

anammox occurrence was investigated(Li et al., 2019). Also, the soluble COD and

76

stable nitrite production might be the two major challenges for large scale applications

77

of PD/A process (Zhang et al., 2019). Although extensive research has been carried

78

out on the treatment of NO3--N -rich synthetic wastewater, to the best of our

79

knowledge, no studies have assessed the leachate treatment process using PD/A to

80

realize enhanced nitrogen removal. To date, the feasibility and mechanisms of

4

81

leachate treatment using the PD/A process remain unclear.

82

Therefore, this study aimed to develop a novel combined process based on PN/A

83

and PD/A, to achieve enhanced nitrogen removal. The PN reactor produced nitrite for

84

the Anammox reactor while the excess nitrate generated by the Anammox reactor was

85

removed via the PD/A reactor.

86

2 Materials and methods

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2.1 Experimental apparatus and operation

88 89

Figure 1 As shown in Fig.1, a novel continuous-flow combined process including a

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continuous flow reactor and two UASB reactors was developed for advanced nitrogen

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removal from mature leachate. The PN reactor was divided equally into eight cells,

92

including one anoxic zone and seven aerobic zones. The anoxic zone was equipped

93

with a mechanical stirrer, with compressed air flushed into the remaining aerobic

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zones under the control of a gas flowmeter. The raw landfill leachate was divided into

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the PN influent tank, the Anammox reactor and the partial denitrification-Anammox

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reactor. The working volume of the A/O, Anammox, and PD-Anammox reactors was

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10.5L, 10L, and 3.5L, respectively. The carriers used in this study were a mixture of

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cylinder polypropylene suspended carriers and cubic sponge carriers. The carriers

99

were fixed together by fish wire and fixed into a plastic rack at a 35% fill ratio of

100

working volume. The SRT of the A/O, Anammox, and PD-Anammox reactors was

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25d, 70d, and 65d, respectively. The HRT of the A/O, Anammox, and PD-Anammox

102

reactors was 33.6h, 26.7h, and 21h, respectively.

5

103 104

2.2 Influent and seeding sludge The landfill leachate was taken from a sanitation landfill site which had been

105

operated for 20 years. The main characteristics of the leachate are shown in Table 1.

106

The seed partial nitrification sludge was collected from a pilot-scale SBR system

107

(working volume: 6.28m3) treating real domestic sewage(Yang et al., 2009).

108

Anammox sludge was inoculated from a lab-scale UASB Anammox reactor treating

109

synthetic wastewater. The inoculated partial-denitrification sludge was collected from

110

a PD/A SBR within our laboratory, which simultaneously removed ammonia and

111

nitrate. Table 1

112 113

2.3. Analytical methods

114

Values of MLSS, MLVSS, ammonium, nitrate, nitrite and COD were measured

115

according to the standard methods (APHA, 1998). pH/Oxi 340i analyzers were used

116

to monitored DO, pH, and the temperature of the reactors. BOD5 was measured by

117

BOD5 analyzer (Lianhua, LH-BOD601, Lanzhou, China).

118

2.4. Calculations

119 120

The efficiency of ammonium, nitrite, nitrate, Total nitrogen and COD removal was calculated according to Eq.(1): Efficiency=(CInf – CEff) ×100% / CInf

(1)

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where Cinf is ammonium (or nitrite, nitrate.) concentration in the influent and Ceff is

122

the one in the effluent.

123

The overall mass balance on nitrogen and COD can be measured as followed:

6

CODinf = CODeff + CODsludge + CODDN + CODO2

(2)

124

Where, CODinf and CODeff are the COD in the influent and effluent, mg/d; CODsludge

125

is the COD converted to biomass, mg/d; CODDN is the COD consumed by

126

denitrification, mg/d; CODO2 is the carbonaceous COD consumed during aeration,

127

mg/d.

128

The COD converted to biomass could be estimated by Eq(3): CODsludge = 1.42 × Csludge × Qsludge

(3)

129

Where Csludge means concentration of disposal sludge, mg/L; Vsludge means flow rate of

130

disposal sludge liquor, L/d; 1.42 means theoretical value of COD/biomass under the

131

condition of C5H7NO2 being considered as biomass.

132

CODDN was estimated via denitrification, which consists of two parts. One is the COD

133

consumption for denitrification of unit NO2--N, and the other is the consumption for

134

denitrification of unit NO3--N (Eq.(4))(Wang et al., 2016a). CODDN = 2.86 × NO3--NRE + 1.71 × NO2--NRE

(4)

135

Where the NO3--NRE and NO2--NRE are the removed mass of NO3--N and NO2--N via

136

denitrification, mg/d . As for CODO2, it is negligible since nearly all the COD in the

137

PN reactor was used for denitrification in the anoxic cell. Also, the mass balance of

138

nitrogen could be established similarly.

139

The NTR was calculated according to Eq.(5): NTR = (NO2--NEff –NO2--NInf) ×100% / (NO3--NInf –NO3--NEff)

140 141

(5)

The nitrogen removal ratio (NRR) was calculated according to Eq.(6) (Miao et al., 2015) :

7

NRR = [(NO3--NInf + NH4+-NInf+ NO2--NInf－NO3--NEff－NH4+-NEff－ (6) --N

NO2 142

Eff)]

×VInf

×10-3/

(V×T)

The FA and FNA concentration was calculated according to pH and temperature

143

and the concentration of ammonium and nitrite (Anthonisen et al., 1976).

144

3 Results and discussion

145

3.1 Performance of the PN, Anammox, and PD/A reactors

146

3.1.1 Performance of the partial nitrification reactor Figure 2

147 148

In this study, the PN reactor produced nitrite for the Anammox process. The

149

nitrogen concentration profiles in the partial nitrification reactor are shown in Fig. 2a

150

and b for the whole operation period. After changing the reflux ratio from 100% in

151

phase 1 to 200% in phase 2, the NH4+-N transformation rate and NO2--N

152

accumulation rate reached 93.4% and 91.5%, respectively. Due to the high leachate

153

concentration of FA (43.5 mg/) and FNA (0.18 mg/L), NOB bacterial activity was

154

suppressed throughout the entire reaction period. As shown in Fig. 2c, the specific

155

ammonia oxidation rate (SAOR) of 3.4-4.2 mgN/gVSS·h was higher than the specific

156

nitrite oxidation rate (SNOR) of 0.6-1.32 mgN/gVSS·h, which also demonstrates the

157

realization of partial nitrification.

158

3.1.2 Performance of the Anammox reactor

159

Figure 3

160

Fig. 3 presents the nitrogen profile in Anammox reactor throughout the whole

161

operational period. A bypass from the influent tank (bypass 1) provided NH4+-N for

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the Anammox process in the UASB reactor. After combining the flow in bypass 1

163

with 38% landfill leachate, the average removal ratios of NO2--N / NH4+-N and

164

NO3--N / NH4+-N were 1.35 and 0.31, respectively. These results were close to

165

the respective stoichiometric ratios obtained by (Jetten et al., 1998; Strous et al.,

166

1998). The average effluent concentrations of NH4+-N and NO2--N were 19.6 mg/L

167

and 11.5 mg/L. Correspondingly, the NRR in the Anammox reactor reached to 1.67

168

kgN/(m3·d). The Anammox reactor contributed 72% of the total nitrogen removal of

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the combined process. Besides, the average effluent concentration of NO3--N

170

increased to 148.3 mg/L, due to the Anammox reaction in the UASB reactor. To meet

171

the requirements of the TN discharge standard and achieve a higher nitrogen removal

172

efficiency, NO3--N must be removed in the subsequent process.

173

3.1.3 Performance of PD-Anammox reactor Figure 4

174 175

In this combined system, the PD-Anammox process was included to remove

176

excess nitrate from the Anammox reactor. The nitrogen profile, COD/NO3--N ratios

177

and bypass Ⅱ ratio in the PD-Anammox reactor are presented in Fig. 4, for the entire

178

120-day operational period. After altering the COD/NO3--N ratio from 1.0 to 4.0 and

179

the bypass Ⅱ ratio from 5% to 3%, TN concentrations in the effluent decreased

180

gradually from 210.1 mg/L to 15.7 mg/L. The removal efficiency of total nitrogen

181

increased to 92% on average in the PD-Anammox reactor. According to Table 2, the

182

average effluent NO3--N concentration was reduced from 116.1 mg/L in phase 1 to

183

4.0 mg/L in phase 4.

9

184

As shown in Table 2, the NTR in the PD-Anammox reactor varied according to a

185

change in COD/NO3--N ratio from 1.0 to 4.0. In previously reported studies, a

186

COD/NO3--N ratio of 3.0 has commonly been selected to achieve maximum

187

accumulation of NO2--N during the partial denitrification process. It is of note, that

188

the NTR of the PD-Anammox reactor in phase 1 was 65.2%, due to the lack of

189

biodegradable COD. Hence, the contribution of Anammox to total TN removal

190

reached 60.3%. After altering the COD/NO3--N ratio from 1.0 to 4.0, the NTR of

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partial denitrification and the contribution of Anammox to total TN removal reached

192

60.4% and 57.1%, respectively. However, the TN removal rate increased from 22.1%

193

(phase 1) to 92.1% (phase 4) due to the synergy of complete denitrification and

194

Anammox processes.

195

Table 2

196

3.2 Performance of the combined PN/A + PD/A system

197

3.2.1 Nutrient removal in the PN/A+PD/A combined process

198

The performance of COD and nitrogen removal in combined system is depicted

199

in Fig 5. Results show that >98% of total nitrogen was removed throughout the

200

PN/A+PD/A combined process, of which 7%, 73%, and 18% were removed in the PN,

201

Anammox and PD/A reactors, respectively. Nitrogen loss in the PN reactor was

202

achieved via the denitrification process by utilizing the small amount of BOD5 present

203

in the raw leachate. Importantly, the application of Anammox effectively reduced the

204

production of excess sludge in comparison to conventional nitration-denitrification

205

processes. In partial denitrification-Anammox reactors treating early landfill leachate,

10

206

the addition of acetate could be replaced by the addition of early leachate, which

207

contains a sufficient supply of BOD5 for partial denitrification.

208 209

Figure 5 Moreover, only 11% of the total COD was removed throughout the 3-stage

210

combined process. The remaining COD in the effluent was the mostly refractory

211

organic matter which cannot efficiently be utilized by heterotrophic bacteria in the

212

combined process. The fluorescence EEM spectra profiles of leachate pre- and

213

post-treatment using the PN/A+PD/A process are also investigated. For raw landfill

214

leachate, the main fluorescent component was aromatic protein-I (Ex < 250nm, Em <

215

330nm). The raw mature leachate contained a higher proportion of protein-like

216

compounds, but these were not detected in the effluent. After treatment using the

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partial nitrification process, the main fluorescent component changed to humic-like

218

acid (Ex > 250 nm, Em > 380 nm), which is supported by the findings reported by

219

previous studies(Chen et al., 2019). Similarities in fluorescent peak locations

220

indicated that the compounds dominantly remaining in the effluent resembled

221

humic-like acid, which existed in leachate but could not be sufficiently utilized by

222

microorganisms. Furthermore, the fluorescence intensity of the humic-like acid signal

223

increased throughout treatment using the Anammox and PD/A reactors. The ultimate

224

removal of remaining fulvic-like acid compounds could require physicochemical

225

treatments, such as ozonation and electrochemical methods(Ye et al., 2016).

226 227

Recently, COD removal via anaerobic processes and the conception of energy-positive wastewater treatment plants, have drawn much research attention.

11

228

COD capture technology has been commonly applied for the treatment of early

229

leachate using an anaerobic reactor. The high concentrations of BOD in leachate was

230

captured in the form of CH4 through high-efficiency anaerobic digestion and used for

231

the co-generation of heat and power. However, the conception of energy-positive

232

sewage treatment plants based on autotrophic nitrogen removal such as Anammox

233

still requires significant development and optimization to overcome the difficulty in

234

achieving a stable supply of NO2--N. Therefore, the realization of stable partial

235

nitrification and partial denitrification in this three-stage process treating mature

236

landfill leachate provides a possibility for the development of industrial-scale

237

energy-positive sewage treatment plants.

238

3.2.2 Significance of the combined PN/A+PD/A process for landfill leachate

239

treatment

240

Conventional secondary treatment of landfill leachate commonly occurs via

241

complete nitration and denitrification, which produces large volumes of excess sludge

242

and requires a high amount of energy for aeration and backflow dilution. The high

243

NH4+-N concentration of leachate (up to 1000mg/L) was diluted to 70-80 mg/L

244

through high ratio reflux from the effluent, providing a high NH4+-N concentration for

245

partial nitrification. It has previously been reported that up to 60% of the operational

246

costs are mainly attributed to electricity requirements and treatment of excess

247

activated sludge. Due to the high ammonia characteristic of landfill leachate, novel

248

leachate treatment processes based on Anammox such as Sharon-Anammox, PN/A

249

and SNAD, have attracted much research attention, due to their advantages of reduced

12

250

costs, energy requirements and use of resources. 68% of TN removal was achieved

251

using combined partial nitrification and Anammox in a 304 m3/d flow reactor treating

252

leachate (Wang et al., 2010). Residual nitrate concentrations of 71mg/L were removed

253

through heterotrophic denitrification, following the addition of 500mg/L COD.

254

Similarly, by using Single stage Nitrogen removal using Anammox and Partial

255

nitritation (SNAP) process dealing with diluted mature leachate, 91.8% TN removal

256

efficiency was achieved. However, the concentration of nitrate achieved 134mg/L in

257

the effluent, which required further treatment to meet the discharge standard (Wen et

258

al., 2016). Compared with complete denitrification processes, the cooperation of

259

partial denitrification and Anammox has been demonstrated to provide a potentially

260

economical alternative method for enhanced nitrogen removal. In the present study,

261

the requirement for an external carbon source was reduced by >42% with an NTR of

262

60%, while the energy costs associated with aeration during nitrification could be

263

reduced by 100%. Moreover, the production of greenhouse gases such as N2O and

264

NO during complete denitrification, are also decreased in the PD/A process(Du et al.,

265

2019). Therefore, this study supports the potential use of the PN/A+PD/A process for

266

practical application in the treatment of landfill leachate, with a high nitrogen removal

267

efficiency.

268

3.3 Microbial community diversity analysis of the PN, Anammox, PD/A-sludge

269

and PD/A-biofilm systems

270 271

In order to profile the microbial community structures in all three reactors, sludge and biofilm samples were collected in the steady phase on day 110. The bacterial

13

272

community at the phylum level dominantly consisted of Proteobacteria, Chloroflexi,

273

Bacteroidetes, Planctomycetes and Deinococcus-Thermus, which is in agreement with

274

previous studies on microbial communities in denitrification reactors (Cao et al., 2016;

275

Ma et al., 2017)and wastewater treatment reactors(Liu et al., 2017). Nitrosomonas

276

(2.6%) and Nitrosomonadaceae (0.17%) are typical ammonia oxidation bacteria

277

(AOB) reported in partial nitrification reactors. Nitrobacter, a known nitrite-oxidizing

278

bacteria (NOB) (Cebron & Garnier, 2005), was well represented in the PN reactor

279

with a relative abundance of 0.94%. The difference between AOB and NOB

280

quantities can likely be explained by the high NAR observed in the PN reactor.

281

In this study, Candidatus Brocadia, Candidatus Kuenenia, and Candidatus

282

Jettenia were detected in Anammox and PD/A-reactor. C. Brocadia (11.5%) was

283

more abundant in the Anammox reactor than with C. Kuenenia (0.8%). In contrast, C.

284

Kuenenia was most abundant Anammox genus in the PD/A reactor, with a biofilm

285

abundance of 3.2% and a sludge abundance of 0.7%. The relative abundance of C.

286

Brocadia in the biofilm and sludge were 1.4% and 0.3%, respectively. Furthermore, C.

287

Jettenia was detected in the PD/A reactor with an abundance of 0.15% in the biofilm

288

and 0.16% in sludge, while being undetected in the Anammox reactor. A possible

289

explanation for the different Anammox microbial communities in the Anammox and

290

PD/A reactors was the higher nitrite affinity of C. Kuenenia than C. Brocadia (Lotti et

291

al., 2014; Yang et al., 2017; Zhang et al., 2017). However, it remains challenging to

292

explain the difference in Anammox microbial communities as ecological niche

293

differentiation of Anammox bacteria remains unresolved.

14

294

Both PD/A-biofilm and PD/A-sludge communities shared several principal

295

functional bacterial genera, such as Thauera, C. Kuenenia, and C. Brocadia. However,

296

the biofilm played a more prominent role in nitrogen removal through Anammox, as

297

compared with the PD/A-sludge. Phylogenetic analysis of samples presented in

298

Figure. 6a reveals the similarity between PD/A-biofilm and Anammox sludge

299

communities. The inconsistency between sludge and biofilm communities in the

300

PD/A-reactor was consistent with previous reports showing that Anammox bacterial

301

communities preferentially secrete extracellular polymeric substances (EPS) and

302

attach to biofilms and pipe walls(Vlaeminck et al., 2010; Wrl et al., 2010). Figure 6

303 304 305

4 Conclusions The novel continuous-flow PN/A+PD/A process for enhanced nitrogen removal

306

from mature landfill leachate was established successfully, achieving a final TN

307

removal efficiency of 98.8% and effluent TN concentration of 15.7mg/L. Detailed

308

analysis of the nitrogen mass flow revealed that only 7% of nitrogen removal

309

occurred in the PN reactor via nitritation-denitritation, together with 73% and 18%

310

removal in the Anammox and PD/A reactors, respectively. Batch tests revealed that

311

the PD/A-biofilm played a more dominant role in nitrogen removal than PD/A-sludge.

312

Candidatus Brocadia and Candidatus Kuenenia were the most abundant genera in the

313

Anammox reactor and PD/A-reactor, respectively.

314 315

Acknowledgments

15

This research was financially supported by National Natural Science Foundation

316 317

of China (51778216) and Beijing Municipal Science & Technology Project

318

(Z181100005518006).

319 320

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Graaf, A.A., Logemann, S., Muyzer, G., van Loosdrecht, M.C.M., Kuenen, J.G. 1998. The

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420

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18

424

Table Captions:

425

Table 1 Characteristics of the raw leachate used in the study

426

Table 2 The nitrogen removal performance and analysis of PD-Anammox reactor

427

under different operational modes

428 429

Figure Captions:

430

Figure 1 Schematic diagram of the Partial Nitrification-Anammox(PN/A) + Partial

431

Denitrification – Anammox(PD/A) reactor

432

Figure 2 Performance of the partial nitrification reactor in the long-term operation: (a)

433

variations of NH4+-N and NH4+-N transfer efficiency; (b) variations of NO3--N,

434

NO2--N and NO2--N accumulation ratio; (c) bacteria activity of AOB and NOB.

435

Figure 3 Performance of Anammox reactor in different stages: (a) variations of

436

NH4+-N and NH4+-N removal efficiency; (b) variations of NO3--N, NO2--N and

437

NO2--N removal efficiency; (c) the ratio of NO2--Nremoved/NH4+-N removed and NO2--N

438

removed/NH4

439

Figure 4 Performance of PD/A process for simultaneous treatment of NO3--N and

440

landfill leachate in long-term operation (a) the ratio of COD / NO3--N; (b) variations

441

of NO3--N, NO2--N and nitrate removal ratio; (c) variations of TN, NH4+-N and TN

442

removal ratio.

443

Figure 5 Nutrient removal analysis: (a) Mass flows of COD and nitrogen in

444

PN/A+PD/A process; (b) Trends of COD and nitrogen in the system

+-N

removed.

19

445

Figure 6 High-throughput sequencing analysis: (a) Community heat map and

446

taxonomic classification at the phylum level; (b) The differences in phylum level

447

between sludge and biofilms in PD/A reactor

448

20

449

Table 1 Characteristics of the raw leachate used in the study Values are in mg/L, except the pH. Compound

Mean

Max

Min

COD

2390

2448

2231

BOD5

238

250

169

NH4

1454

1646

1355

NO3-N

2.3

4.1

3

NO2-N

0.3

1.5

0

TN

1570

1849

1021

Alkalinity

12470

13530

11300

pH

8.3

8.6

8.0

TP

4.5

7.8

3.2

+-N

450

21

451

Table 2 The nitrogen removal performance and analysis of PD-Anammox reactor

452

under different operational modes Phase (day)

1 (1-30)

2 (31-60)

3 (61-87)

4 (88-123)

Bypass Ⅱ ratio(%)

5

5

3

3

COD/NO3--N

1

2

3

4

Inf

96.3

92.9

61.2

59.6

Eff

78.0

55.7

21.2

9.8

Inf

142.8

126.8

136.5

111.9

Eff

116.1

64.8

51.7

4.0

Inf

23.4

12.4

12.3

23.6

Eff

15.9

7.1

4.7

3.4

Inf

270.2

238.9

217.3

201.1

Eff

210.1

119.5

42.7

15.7

Anammox

60.3

62.6

59.4

57.1

Denitrification

39.7

38.4

40.6

42.9

NTR

65.2

68.2

64.7.

60.4

NH4+-N

NO3--N

NO2--N

TN

Percentage (%) 453

22

454 455

Figure 1 Schematic diagram of the Partial Nitrification-Anammox(PN/A) + Partial

456

Denitrification-Anammox(PD/A) reactor

23

NTE

Phase 2

Phase 1

100

1500

80

1200

60

400

40

200

20

0

(b)

0

Inf. NO2--N，Eff. NO2--N/ (mg/L)

1000

20 Inf. NO2--N

40 Eff. NO2--N

100 80 60 Eff. NO3--N Inf. NO3--N

Phase 1

Phase 2

0 120 NAR

100

800

80

600

60

400

40

200

20

Bacteria activity (mgN/(h·gVSS))

(c)

457

Eff. NH4+-N

0

0

20

40

60

80

AOB

6

NH4+-N transfer efficiency/(%)

Inf. NH4+-N

1800

100

0 120

Nitrite accumulation ratio / (%)

Inf. NH4+-N，Eff. NH4+-N/ (mg/L)

(a)

NOB

4 2 0

0

20

40

60 Time(d)

80

100

120

458

Figure 2 Performance of the partial nitrification reactor in the long-term operation: (a)

459

variations of NH4+-N and NH4+-N transfer efficiency; (b) variations of NO3--N,

460

NO2--N and NO2--N accumulation ratio; (c) bacteria activity of AOB and NOB.

461

24

NH4+-N removal efficiency

Phase 1

Phase 2

100 80

400

60 40

200

20 0

0 Inf. NO2--N 800

20 Eff. NO2--N

(b)

Inf. NO2--N，Eff. NO2--N/ (mg/L)

Eff. NH4+-N

40 60 80 Eff. NO3--N Inf. NO3--N

Phase 1

0 100 120 Nitrite removal efficiency

Phase 2

100

600

80 60

400

40 200

20

0

(c) 2.5

0

20

40 60 80 100 + ▲NO2 -N / ▲NH4 -N ▲NO3--N / ▲NH4+-N

Ratio

2.0 1.5 0.5

462

0 120

1.32

1.0 0.0

NH4+-N removal efficiency/(%)

Inf. NH4+-N

600

Nitrite removal efficiency / (%)

Inf. NH4+-N，Eff. NH4+-N/ (mg/L)

(a)

0.26 0

20

40

60 Time(d)

80

100

120

463

Figure 3 Performance of Anammox reactor in different stages: (a) variations of

464

NH4+-N and NH4+-N removal efficiency; (b) variations of NO3--N, NO2--N and

465

NO2--N removal efficiency; (c) the ratio of NO2--Nremoved/NH4+-N removed and NO2--N

466

removed/NH4

+-N

removed.

467 468

25

9 6

2

3 2

200

(b)

20

2

Eff. NO N

Phase 1

40

60

3

Inf. NO N

Phase 2

80

3

Eff. NO N

100

Nitrate removal ratio

Phase 4

Phase 3

150

60

-

-

100 80

100

40 50

20

0 0

20 Inf. NH4+-N

(c) 400

Phase 1

40 Eff. NH4+-N

60

80

Inf. TN

Eff. TN

Phase 2

100 Phase 4

Phase 3

0 120

TN removal ratio

100 80

300 NH4+-N, TN/ (mg/L)

0 120

60 200 40 100

0

20

0

20

40

60 Time/(d)

80

Nitrate removal ratio / (%)

0

Inf. NO N

469

12 Reflux ratio / (%)

Phase 4

Phase 3

4

0

Inf. NO2 -N，Eff. NO2 -N/ (mg/L)

Phase 2

Phase 1

100

TN removal ratio / (%)

-

COD/NO3 -N

(a) 6

0 120

470

Figure 4 Performance of PD/A reactor for simultaneous treatment of NO3--N and

471

landfill leachate in long-term operation (a) the ratio of COD / NO3--N; (b) variations

472

of NO3--N, NO2--N and nitrate removal ratio; (c) variations of TN, NH4+-N and TN

473

removal ratio

474

26

475

(b)

COD

3000

NH4+-N

NO2--N

NO3--N

1600

TN

COD/(mg/L)

1400 2000 600 1000 300 0

477

Influent

PN-effluent

PD/A-influent

Effluent

0

Sample position

478

Figure 5 Nutrient removal analysis: (a) Mass flows of COD and nitrogen in

479

PN/A+PD/A process; (b) Trends of COD and nitrogen in the system

480

27

NH4+-N, NO2--N, NO3--N, TN/ (mg/L)

476

481

482 483

Figure 6 High-throughput sequencing analysis: (a) Community heat map and

484

taxonomic classification at the phylum level; (b) The differences in phylum level

485

between sludge and biofilms in PD/A reactor

28

486

Author Contributions Statement

487 488

Zhong Wang: Conceptualization, Formal analysis, Investigation, Data curation,

489

Writing - original draft

490

Liang Zhang: Validation, Writing - review & editing

491

Fangzhai Zhang: Project administration

492

Hao Jiang: Investigation

493

Shang Ren: Sampling and analysis

494

Wei Wang: Resources

495

Yongzhen Peng: Supervision, Funding acquisition, Writing - review & editing

496 497 498 499

Declaration of interests

500

☒ The authors declare that they have no known competing financial interests or

501 502

personal relationships that could have appeared to influence the work reported in this paper.

503 504 505 506

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

507

29

508 509

Highlights

510

 A continuous-flow process based on PN/A and PD/A was developed to treat

511 512 513

leachate.  NH4+-N conversion rate of 93.4% and NAR of 91.5% were realized in A/O reactor.

514

 The remaining DOM in effluent was mainly fulvic-like substances.

515

 The nitrate-to-nitrite transform ratio (NTR) reached to 60.4% in PD/A reactor.

516

 The effluent TN of 15.7mg/L and TN removal efficiency of 98.8% were

517

achieved.

518 519

Graphic abstract

30

520 521

31