Coupled sulfur and iron(II) carbonate-driven autotrophic denitrification

Subscriber access provided by Iowa State University | Library

Remediation and Control Technologies

Coupled sulfur and iron(II) carbonate-driven autotrophic denitrification for significantly enhanced nitrate removal Tingting Zhu, Hao-Yi Cheng, Lihui Yang, Shigang Su, Hongcheng Wang, Shusen Wang, and Ai-Jie Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06865 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Environmental Science & Technology

1

Coupled sulfur and iron(II) carbonate-driven autotrophic

2

denitrification for significantly enhanced nitrate removal

3

Tingting Zhu a , Hao-yi Cheng a *, Lihui Yang a,b, Shigang Su a,b, Hongcheng Wang a,b,

4

Shusen Wang a,b, Ai-jie Wang a,b, *

5 6

a.Key Laboratory of Environmental Biotechnology, Research Center for Eco-

7

Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, P. R. China.

8

b.University of Chinese Academy of Sciences, Beijing, 100049, P. R. China.

9

*Corresponding author: Key Laboratory of Environmental Biotechnology, Research

10

Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

11

100085, P.R. China; E-mail address: [email protected] (Ai-Jie Wang) &

12

[email protected] (Hao-Yi Cheng).

13

ACS Paragon Plus Environment


15

Abstract

16

Sulfur-based denitrification process has attracted increasing attentions because it

17

does not rely on the external addition of organics and avoids the risk of COD exceeding

18

the limit. Traditionally, limestone is commonly employed to maintain a neutral

19

condition (SLAD process), but it may reduce the efficiency as the occupied zone by

20

limestone cannot directly contribute to the denitrification. In this study, we propose a

21

novel sulfur-based denitrification process by coupling with iron(II) carbonate ore

22

(SICAD system). The ore was demonstrated to play roles as the buffer agent and

23

additional electron donor. Moreover, the acid produced through sulfur driven

24

denitrification was found to promote the Fe(II) leaching from the ore and likely extend

25

the reaction zone from the surface to the liquid. As a result, more biomass was

26

accumulated in the SICAD system compared with the controls (sulfur, iron(II)

27

carbonate ore and SLAD systems). Owing to these synergistic effects of sulfur and

28

iron(II) carbonate on denitrification, SICAD system showed much higher

29

denitrification rate (up to 720.35 g∙N/m3∙d) and less accumulation of intermediates

30

(NO2- and N2O) than the controls. Additionally, sulfate production in SICAD system

31

was reduced. These findings offer great potential of SICAD system for practical use as

32

a highly efficient post-denitrification process.


Page 2 of 32

Page 3 of 32


34

1. Introduction

35

Heterotrophic denitrification is the conventional and most widely adopted method

36

for the treatment of secondary effluent. However, this process requires carbon sources

37

as electron donors1, which increases the operational cost2 and leads secondary pollution.

38

The accurate addition of the carbon source is difficult3. Therefore, organic independent-

39

autotrophic denitrification driven by hydrogen, sulfur or reduced iron species has

40

gained increasing attentions4. Thereinto, element sulfur driven denitrification is relative

41

highly efficient and holds the advantages of cost-effective and safety. The overall

42

reaction for sulfur-based autotrophic denitrification is described as in Eqs (1), which

43

can be seen as an acid producing process.

44

55S0+50NO3-+38H2O+20CO2+4NH4+ →4C5H7O2N+55SO42-+25N2+64H+

45

In practice, limestone is commonly employed into the sulfur based denitrification

46

system. It serves as a pH buffering agent and the source of inorganic carbon for bacteria

47

growth5, which is known as sulfur-limestone autotrophic denitrification (SLAD).

48

Although limestone is an effective external alkalinity source, it cannot play a role as

49

electron donor. Therefore, the occupied zone by limestone will not contribute to the

50

denitrification and may limit the efficiency of the system. Additionally, as shown in

51

Eqs (1), sulfate can be produced during the denitrification (7.54 mg SO42- per mg NO3--

52

N), which should be controlled in case of meeting certain standards. For example, the

53

sulfate limit in drinking water standard is 250 mg/L in China. That means the permit

54

concentration of NO3--N in the target treating water cannot over 33 mg/L. If there is

55

some background value of sulfate in the water, the permit concentration will be even

56

lower. This may limit the application of this technology.

(1)

57

Nitrate-dependent anaerobic ferrous oxidation (NAFO) process has been

58

intensively explored using Fe(II) as an electron donor to convert nitrate into nitrogen

59

gas under anoxic conditions6-8. Iron(II) carbonate (FeCO3)9-11 is one of the most

60

abundant Fe(II)-bearing minerals in nature and plays an important role in iron and

61

carbon cycling. Recent research12 found that iron(II) carbonate ore (siderite, FeCO3 as

62

the main component) can also drive the conversion of nitrate to nitrogen gas. The

63

overall reaction of denitrification driven by iron(II) carbonate is described as



64 65

Page 4 of 32

follows11,12: 5FeCO3+NO3-+8H2O → 5Fe(OH)3+0.5N2+4CO2+HCO3-

(2)

66

This process produces alkalinity and carbonate, which could act as pH buffer agent

67

and the inorganic carbon sources for bacterial growth12. If the iron(II) carbonate was

68

introduced into the sulfur driven denitrification system, it likely plays a similar role as

69

the limestone but avoids the increase of hardness. In addition to the function of buffer

70

agent13, iron(II) carbonate ore can work as an additional electron donor6,14-17 and

71

directly contribute to the denitrification. For this reason, the denitrification efficiency

72

is expected to be higher than the conventional SLAD process in which the occupation

73

of limestone can forms an inactive zone of denitrification in the system. Another

74

expected advantage is the decline of the sulfate production, as partial nitrate removal

75

will follow the iron(II) driven denitrification route. To our best knowledge, the

76

combination of element sulfur and iron(II) carbonate ore to enhance the denitrification

77

efficiency has not been reported yet.

78

In this study, a sulfur-iron(II) carbonate autotrophic denitrification (SICAD)

79

system was investigated in reactors packed with element sulfur and siderite particles.

80

The denitrification performance of SICAD system was studied by continuously feeding

81

nitrate containing solution at different hydraulic retention time (HRT) conditions and

82

compared with SLAD, sulfur alone, and iron(II) carbonate alone systems in terms of

83

nitrate removal rate, N intermediates distribution and pH. The measurement of Fe(II)

84

leaching and mössbauer analysis were performed to explore the roles and the fate of

85

iron in the system. High-throughput 16S rRNA gene based Illumina MiSeq sequencing

86

were employed to understand the microbial communities in different systems. The main

87

objective of this study are (i) to verify the SICAD system has superior denitrification

88

performance compared to the SLAD, sulfur alone and iron(II) carbonate alone systems

89

and (ii) to reveal the possible synergistic effects of sulfur and iron(II) carbonate that

90

result in a high denitrification efficiency in the SICAD system.

91

2. Materials and methods

92

2.1 Reactor setup

93

Sulfur (radius 1.5 to 3.5 mm) was packed into an up-flow column reactor (radius


Page 5 of 32


94

10.5 cm × 30 cm) with a working volume of 1 L (hereafter referred to as SAD system)

95

by connecting a gas-tight bag. Iron(II) carbonate (radius 1.0 to 1.5 mm), a mixture of

96

sulfur with iron(II) carbonate and a mixture of sulfur with limestone (radius 2.0 to 4.0

97

mm) were also packed into up-flow column reactors (hereafter referred to as ICAD

98

system, SICAD system and SLAD system, respectively). The volume ratio of the

99

mixture was 1:1 and the packed volume of particles was 0.5 L in these four systems.

100

Sulfur came from a chemical plant (ShanDong, China), and the sulfur content was

101

98%±0.5%. Natural iron(II) carbonate was acquired from a mine (GuiZhou, China),

102

and the ferrous content was 86%±0.5%. The limestone came from Sinopharm shares,

103

and the purity was 98%±0.2%.

104

2.2 Inoculation and operation

105

The systems were inoculated with activated sludge (50 ml, 10 g TSS/L) from Gao

106

Beidian sewage treatment plant (Beijing, China) and autotrophic denitrification

107

bacterial (20 mL, 17 g TSS/L) from previous sulfur packed-denitrifying bed reactors,

108

which had been operated for 3 months. The synthetic wastewater was as follows (g/L):

109

NaNO3 0.121, Na2S2O3 1.5, NaHCO3 0.1, NH4Cl 0.002, Na2HPO4 0.15, MgCl2·7H2O

110

0.5, CaCl2·2H2O 0.01 and Wolfe’s trace element solution14 (1 ml/L). The systems were

111

recirculated to allow biofilm establishment. After inoculating for 15 days, the systems

112

were fed in continuous mode under hydraulic retention time (HRT) of 24 h. In the

113

influent, the Na2S2O3 and NH4Cl were removed, and the concentration of NaHCO3 is

114

0.02 g/L. When the stable performance of nitrate removal was achieved, the systems

115

were operated continuously with HRTs from 12 h to 0.5 h to evaluate the effect of

116

SICAD on the denitrification performance. The pH of the influent was controlled at

117

7.3±0.2 by adding 1 M HCl or NaOH solutions. The dissolved oxygen (DO)

118

concentration in the feed was always maintained at less than 0.5 mg-O2/L and the

119

systems were operated in a temperature controlled laboratory, approximately 30±5oC.

120

2.3 Analytical methods

121

The samples were collected through a 0.45-μm membrane filter. Anions (nitrite,

122

nitrate, sulfite and sulfate) were measured with an ion chromatograph (883 Basic IC

123

plus, Metrohm, Swizerland) equipped with a Metrosep A Supp 5-250 column (Metrohm,



Page 6 of 32

124

Swizerland). The concentration of N2O was determined using gas chromatography-

125

mass spectrometry as described in Cheng et al15. The N2O concentration in the liquid

126

phase was calculated according to Henry's law. The concentration of Fe2+ and sulfide

127

were measured regularly according to standard methods16. The total Fe concentration

128

was determined by inductively coupled-optical emission spectroscopy (ICP-OES,

129

PerkinElmer-OPTIMA 8300). The free nitrous acid (FNA) concentration was

130

calculated

131

Ka=5.05×10-4 at 30.0oC (Ka=e-2300/T, T in degrees Kelvin)17.

132

2.4 The X-ray diffraction analysis (XRD)

based

on

the

relationship

[FNA]=46/14×[NO2-]/(Ka×10pH)

with

and Mössbauer spectroscopy

133

The composition of iron(II) carbonate was determined by XRD analysis (X'Pert

134

PRO MPD, PANalytical B.V., Netherlands) using CuKα radiation, which was shown

135

in Figure S1. The composition of the precipitate that formed on the iron carbonate was

136

determined by Mössbauer spectroscopy (WissEl-WA-260) at 78 K. Samples were taken

137

from the surface of the iron(II) carbonate in SICAD system and washed with deionized

138

water. Then, the samples were percolated through a 0.22-μm membrane filter after

139

centrifugation. To minimize oxidation by O2, the treated membrane was sealed between

140

two layers of Kapton tape under anaerobic conditions. The sample detection was

141

identical to the procedures reported in Peretyazhko et al18. The data analysis procedures

142

for Mössbauer spectra followed a report by Morris et al19.

143

2.5 Scanning electron microscopy (SEM)

144

SEM images were obtained using a SU-8020 scanning microscope (Hitachi, Japan)

145

to get additional visual insight into the microorganisms on the particle surface. Particles

146

were randomly collected from four systems, and put in a tube, respectively. The sample

147

preparation was identical to the procedures reported in Wang et al20, which was

148

presented in the Supplemental Material.

149

2.6 Microbial community analysis and adenosine triphosphate (ATP)

150

The samples for the microbial community analysis were taken at the end of

151

experiment. Particles from the top, middle, and bottom sections of each system were

152

pre-treated as described previously21. It was performed in triplicate. The collected

153

mixture of particles were taken for total genomic DNA extraction using the protocol of


Page 7 of 32


154

the FastDNA SPIN Kit for soil (Sangon Biotech Co., Ltd, Shanghai, China) according

155

to the manufacturer’s instructions. All extracted DNA were stored at -80oC for further

156

amplifications. The primers 515F (5’-GTGCCAGCMGCCGCGG-3’) and 806R (5’-

157

GGACTACHVGGGTWTCTAAT-3’) which target V4 hypervariable regions of

158

microorganism 16S rRNA genes were selected22, and tagged with paired barcode

159

sequence (12 mer) for pooling of multiple samples in one sequencing run. The details

160

for 16S rRNA gene PCR amplification are available in the Supplemental Material. All

161

primers were synthesized by Sangon Biotech (Shanghai, China).The raw sequencing

162

data were generated with a MiSeq sequencing machine in fastq format. All sequencings

163

were clustered at a 97% similarity level, and taxonomic assignments were made online

164

by analysing the MiSeq data on the Usearch 7.0 platform (http://drive5.com/uparse/).

165

The beta diversity of the microbial communities was described using the Principal

166

coordinate analysis (PCoA) and heatmap analysis. The raw sequence files were

167

submitted to the NCBI Sequence Read Archive database and assigned accession No.

168

SRP172574.

169

Filters (i.e. sulfur and siderite particles) were collected from top, middle and

170

bottom sites of each system. The samples of three different parts were mixed for ATP

171

and protein analysis. All ATP and protein measurements were performed in triplicate,

172

and procedures were performed under sterile conditions. The collected particles were

173

soaked in 10 ml sterile water homogenized by vortexing for 5 min at full speed to wash

174

the bacterial on the particles. Then the solution was installed in a new tube and

175

centrifuged at 8000r for 10 min and remove the supernatant. ATP was measured using

176

the BacTiter-GloTM Microbial Cell Viability Assay (G8231, Promega Corporation,

177

Dübendorf, CH) and a GloMax 20/20 Luminometer (Turner BioSystems, Sunnyvale,

178

CA, USA). We applied the optimized protocol described by Hammes et al23 for ATP

179

analysis, comparing with the manufacture’s protocol for this product. Total ATP was

180

measured as follows Zhang et al24, which was added in the Supplemental Material. A

181

conversion factor of 1.75×10-10 nmol ATP per cell was applied to calculate the bacterial

182

cell number24, 25. Protein analysis was performed as the methods of ATP pre-treatment.

183

Then the protocol of the Nuclear and Cytoplasmic Protein Extraction Kit (Sangon



184

Biotech Co., Ltd, Shanghai, China) according to the manufacturer’s instructions was

185

using for protein analysis, described previously by Zhou26.

186

3. Results

187

3.1 The nitrate removal performance

188

To compare the nitrate removal performances among different autotrophic

189

denitrification systems, artificial wastewater contained 20 mg/L of NO3--N was input

190

to all of these systems as influent for treating. As shown in Figure 1 (A), the nitrate

191

removal performance in ICAD system is worse, which was 72.29% and 40.85% at

192

longer HRT of 12 h and 6 h, respectively. The nitrate removal performance ranged from

193

91.31% to 97.42% in SAD, SICAD and SLAD system, respectively. The difference

194

among these three systems of nitrate removal performance seemed minor. When the

195

HRT was decreased to or even less 3 h, the SICAD system had an obviously better

196

performance on nitrate removal than other systems. At a HRT of 3 h, the nitrate removal

197

efficiency in the SICAD system was 92.6±2.5%, which was much higher than those in

198

SAD system (61.0±3.2%), ICAD system (21.9±4.9%) and SLAD system (74.3±4.4%).

199

It was apparent that the existence of both sulfur and iron(II) carbonate helped SICAD

200

system to resist the negative shock of decreasing HRT (from 3 h to 0.5 h) compared

201

with the other systems. At a shorter HRT of 0.5 h, the nitrate removal rate in SICAD

202

system was 80.3±3.9%, which was higher than that in SAD system (33.2±3.8%), IAD

203

system (10.9±5.2%) and SLAD system (56.1±5.1%).

204 205

Figure 1 3.2 Total nitrogen (TN) removal and intermediate accumulation

206

To further investigate the denitrification process in different systems, the TN

207

removal performance was shown in Figure 1 (B). The biological denitrification is an

208

effective approach to remove nitrate (NO3--N), where NO3--N serves as an electron

209

acceptor and is sequentially reduced to nitrite (NO2--N), nitrous oxide (N2O) and

210

nitrogen gas (N2). Due to the absence of NH4+-N in the artificial influent, organic

211

nitrogen and hydroxylamine were excluded. Hence, calculated total nitrogen (TN)

212

included NO3--N, NO2--N and N2O. The TN removal performance in the SICAD system

213

(Figure 1 (B)) was higher than that in the other systems at different HRTs. At a HRT of


Page 8 of 32

Page 9 of 32


214

0.5 h, the TN removal rate in the SICAD system was 720.35 g∙N/m3∙d. It was a 3.30-

215

fold improvement over that in SAD system, 11.30-fold and 1.50-fold higher than that

216

in ICAD system and SLAD system, respectively. The intermediates produced during

217

the denitrification process were studied as well, which was shown as the form of

218

nitrogen balance in Figure 2. At a HRT of 0.5 h, the ratio of intermediates accumulation

219

to total nitrogen was 2.06% in SICAD system, 10.86% in sulfur system, 4.10% in

220

iron(II) carbonate system and 7.15% in SLAD system. This suggested that the SICAD

221

system had the lowest accumulation of intermediates and performed the highest TN

222

removal.

223

Figure 2

224

As shown in Table 1, the produced NO2--N and soluble N2O during denitrification

225

process in different systems were investigated. The effect of different particle (i.e.

226

sulfur and iron(II) carbonate) on the NO2--N and N2O accumulation during the

227

denitrification process were significant. The N2O concentration in the SICAD systems

228

was the lowest, ranging from 0 to 0.006 mg/L. In comparison, it was 130-folder higher

229

in SAD system, 2.5-folder higher in ICAD system and 5-folder higher in the SLAD

230

system, respectively. It illustrated that N2O release could be reduced by SICAD. In

231

addition, the accumulated nitrite in the SICAD system was the lowest as well, ranging

232

from 0 to 0.41 mg/L, while it increased 7.24-fold in SAD system, 2.00-fold in the ICAD

233

system and 3.41-fold in the SLAD system, respectively. It suggested that SICAD could

234

also reduce the NO2- accumulation. Table 1

235 236

3.3 Sulfate production

237

As shown in Figure 3 (A), the sulfate production during per nitrate removal in

238

SICAD system was less than that in SAD and SLAD systems. The sulfate production

239

ranged from 5.67 to 6.92 g/gN in SICAD system, 6.15 to 7.92 g/gN in SAD system and

240

6.78 to 7.98 g/gN in SLAD system. Sulfite and sulfide had not been detected during the

241

entire denitrification process. It was shown that the SICAD system could decrease

242

sulfate production during per nitrate removal, which was helpful to control the sulfate

243

release to the water considering the sulfate limit of 250 mg/L for drinking water. This



Page 10 of 32

244

was attributed to a portion of the nitrate was involved to the NAFO process, resulting

245

in a lower sulfate production than sulfur-based denitrification. The produced sulfate in

246

SAD and SLAD system was lower than expectation based on the reaction stoichiometry

247

(1) with most of the applied HRTs (ie. 12, 1.5 and 0.5 h). This result can be attributed

248

to the incomplete denitrification, as reported in other researches27. The reduction of

249

nitrate in the presence of sulfur can be represented as reactions (6) depending on

250

whether the end product is nitrogen gas or nitrite:

251

S0+3NO3-+H2O→SO42-+3NO2-+2H+

252

S0+2NO2-→SO42-+N2

253 254

(6) Figure 3

3.4 The fate of iron(II) carbonate in SICAD and ICAD systems

255

To investigate the contributions of iron(II) carbonate on the autotrophic

256

denitrification, the fate of iron(II) carbonate was investigated. It was seen that SICAD

257

promoted ferrous leaching from iron(II) carbonate. This result was consistent with the

258

highest nitrate removal in SICAD system, as shown in Figure 3 (B). At a HRT of 0.5 h,

259

the ferrous leaching was 0.86 mg/L in SICAD system, which was higher than that in

260

ICAD system (0.14 mg/L). Based on the sulfate production and ferrous leaching, the

261

contributions of iron(II) carbonate and sulfur on nitrate removal in SICAD system were

262

calculated, as shown in Table S1. At a HRT of 0.5 h, the contribution of iron(II)

263

carbonate and sulfur on autotrophic denitrification was 42% and 58%, respectively. The

264

contribution of iron(II) carbonate increased from 8% to 42% along with the decrease of

265

HRT from 12 h to 0.5 h. Shen and Zhao et al 11,13 studied that the iron(II) products of

266

NADF process were ferric compounds, such as Fe(OH)3. As shown in Figure 3 (B), the

267

concentration of ferric compounds was the difference between the total iron and ferric

268

leaching, which was small. The total iron leaching in SICAD system was higher than

269

Fe(II) by 0.0087 mg/L at HRT of 0.5 h. It indicated that ferric compounds in the effluent

270

of SICAD system was low, which might be attributed to that a part of the produced

271

iron(III) was absorbed on the surface of particles.

272

To further investigate the fate of iron(II) carbonate, Mössbauer spectroscopy was

273

used to analyze the surface morphology of the iron(II) carbonate particles. The relative


Page 11 of 32


274

proportions of the areas covered by doublets and sextets to the total area of the

275

Mössbauer spectrum were equivalent to the relative abundance of Fe in a particular

276

crystal lattice site to that of the total Fe. The iron carbonate contained only 10% of

277

high-spin Fe(II). The other 90% of the Fe in this sample was either high-spin Fe(III)

278

or low-spin Fe(II), as identified by isomer shift. The aggregates produced on the

279

surface of iron(II) carbonate particles in SICAD system did not show reflections and

280

had a low blocking temperature of 77 K in the Mössbauer spectrum (Figure 3 (C)).

281

This result was in agreement with that was the characteristic of ferrihydrite (88.75)

282

and Ferroalluaudite (11.25%), which is [Fe2+Fe3+2(PO4)2(OH)2·8(H2O)]28.

283

3.6 Microbiology analysis

284

The SEM images revealed that visible microbes formed on the surfaces of particles

285

at these four systems in Figure S2. In order to further study the bacteria attached on

286

the particles, the ATP and protein on the particles in the four systems were listed in

287

Table 2. The bacterial cell numbers were derived from the ATP measurements29, 30.

288

The ATP content was 7.11×108 cell/mm2 on sulfur particles in SICAD system, which

289

was 9.11-fold and 4.08-fold higher than that in SAD and SLAD systems, respectively.

290

The ATP content was 0.52×108 cell/mm2 on iron(II) carbonate particles in SICAD

291

system, which was 13-fold higher than that in ICAD system. This suggested that the

292

bacterial cell numbers were highest in SICAD system compared with the other systems.

293

The results of biofilm production were consistent with the nitrate removal

294

performance, as shown in Figure S3. Bacterial carbon storage in polymers has been

295

previously reported to represent 34-65% of the total biomass31. Hence, the protein

296

content was quantified by the biomass, which was shown in Table 2. The protein

297

content on the sulfur particles and iron(II) carbonate were 0.425 mg/mm2 and 0.476

298

mg/mm2 in SICAD system, while the protein in SAD, ICAD and on the surface of

299

sulfur in SLAD system was 0.12, 0.025 and 0.263 mg/mm2, respectively. It

300

demonstrated that SICAD improved the biomass production in order to enhance the

301

nitrate removal.

302

Table 2

303

Regarding microbial community structure, the main difference at the phylum level



304

among the five particles (i.e. sulfur-particles in SAD, SICAD and SLAD system, iron(II)

305

carbonate-particles in ICAD and SICAD) was the phylum Proteobateria,

306

Ignavibacteriae, Chlorobi, Chloroflexi and Bacteroidetes. Proteobacteria (72.32%)

307

and Chlorobi (9.26%) were the dominant groups on the sulfur-particles in SICAD

308

system, whereas the decreases of Proteobateria (63.92%, 64.82% and 56.71%) and

309

Chlorobi (7.71%, 4.52% and 31.31%) abundances were observed in SAD, ICAD and

310

SLAD systems, respectively.

311

Figure 4

312

At the genus level, the main Proteobacteria composition was Thiobacillus, which

313

had a higher relative abundance (approximately 44.74%) in SAD system than that in

314

other systems (Figure 4 (B)). Thiobacillus are species of autotrophs that could use sulfur

315

as electron donor. As expected, the high abundance of Thiobacillus in sulfur system

316

was consistent with previous research on sulfur-based bioreactor. Chlorobaculum were

317

reported to oxidize sulfide and elemental sulfur, generating sulfate as the terminal

318

oxidation product32. The abundance of Chlorobaculum reached 31.16% in SLAD

319

system, which was higher than that in SICAD (8.68%) and SAD systems (7.71%). The

320

results illustrate that Thiobacillus and Chlorobaculum were the dominant bacteria for

321

nitrate removal in SLAD system. In SICAD system, Thiobacillus (26.42% on the sulfur

322

particles and 34.63% on the iron(II) carbonate particles, p＜0.01) and Ferritrophicum

323

(9.20% on the sulfur particles and 7.56% on the iron(II) carbonate particles, p＜0.01)

324

were the dominant groups. Some researchers found that some kinds of Thiobacillus also

325

could use iron(II) as electron donor6,33. Therefore, these Thiobacillus that could use

326

sulfur and iron(II) might play an important role in SICAD system. Ferritrophicum, a

327

neutrophilic ferrous oxidizing bacteria (FeOB), had been reported to oxidize iron(II) to

328

iron(III) using nitrate as an electron acceptor under anoxic conditions34,35. In addition

329

to Fe2+, FeOB can use H2 and sulfide as electron donors and nitrate can serve as an

330

electron acceptor36,37. It suggested that the Ferritrophicum was responsible for nitrate

331

reduction.

332

Furthermore, the known iron-reducing bacterial genera Geothrix6,38 (p ＜ 0.01)

333

were only found in SICAD and ICAD systems. It accounted for 6.12% and 6.38% on


Page 12 of 32

Page 13 of 32


334

the sulfur particles and iron(II) carbonate particles in SICAD system. It might be

335

important for the ferrous and ferric cycle. Microbial community structures were

336

different from each other at different reactors based on the heatmap analysis (Figure 4

337

(C)) and the PCoA analysis (Figure 4 (D)).

338

4. Discussion

339

4.1 Possible mechanism of enhanced nitrate removal efficiency in SICAD system

340

In this study, the enhanced nitrate removal by coupling sulfur with iron(II)

341

carbonate was verified (Figure 5). Firstly, the nitrate removal improvement might be

342

the synergistic influence of SICAD on reaction process and biomass. Some

343

researchers6,34 studied the FeOB could use iron(II) carbonate ore as electron donor for

344

denitrification. The low solubility of minerals might affect the nitrate removal40-42, but

345

the electron transfer rate between FeOB and the minerals also might result in the lower

346

nitrate removal rate43 Rakshit et al.43 found that a homogeneous reaction involving

347

dissolved Fe(II) in equilibrium with the solid Fe(II) and NO2- could occur in addition to

348

the heterogeneous reaction with solid Fe(II). In this study, the acid produced by sulfur

349

denitrification might improve the release of Fe2+ and extend the reaction zone for the

350

bacteria on iron(II) carbonate from a solid phase to a solid-liquid phase (Figure 5). It

351

was verified by the higher ferrous leaching in SICAD system. Additionally, the report

352

by Straub44 about nitrate-dependent Fe(II) oxidation observed that Thiobacillus

353

denitrificans could couple denitrification with anaerobic oxidation of Fe(II) in common

354

minerals. Thus, Thiobacillus denitrificans could be used for sulfur-based denitrification

355

and ferrous iron-based denitrification. However, Thiobacillus denitrificans cannot be

356

enriched without an additional electron donor6. In addition, the kinetics of sulfur

357

denitrification was limited by electron transfer through the sulfur oxidizing bacteria in

358

direct contact with sulfur or soluble sulfur species45. Therefore, sulfur might act as an

359

electron donor to promote the growth of FeOB in SICAD system. Iron(II) carbonate

360

might promote the electron transfer by enrichment of FeOB in order to enhance the

361

nitrogen removal efficiency. This conclusion was verified by the higher biomass

362

production in SICAD system than other control systems (Table 2).Therefore, the

363

synergistic influence of SICAD on extend the reaction zone and higher biomass led to



364

a much higher nitrate removal rate in SICAD system than other control systems.

365

Secondly, the improved nitrate removal could be resulted from the difference in

366

the pH between SICAD (6.80±0.1 to 7.24±0.1) and other systems. The optimum pH for

367

the dominant bacteria of Thiobacillus is 6.8-7.446. The pH ranged from 6.80 to 7.24 in

368

SICAD system. While the pH was not controlled, it was acidic (6.2±0.1 to 6.7±0.1) in

369

SAD system and alkaline (7.40±0.2 to 7.80±0.2) in ICAD system. The lower nitrate

370

removal was attributed to the unsuitable pH, because most biological activity of

371

denitrifiers was repressed at acidic or alkaline conditions. Although the pH ranged from

372

7.23±0.2 to 7.60±0.3 in SLAD system, limestone could not provide larger reaction

373

surface for denitrifiers as electron donor, compared with iron(II) carbonate in SICAD

374

system. It was consistent with the ATP and protein results (Table 2). The ATP and

375

protein of iron(II) carbonate particles in SICAD system was higher than that of

376

limestone particles in SLAD system.

377

Figure 5

378

Additionally, the redox potential of the S0/SO42- (-0.35 mv)47 couple is lower than

379

the Fe(II)/Fe(III) (+200 mv)48 couple in standard conditions47. It indicated that sulfur

380

might oxidize Fe(III) to Fe(II) in thermodynamically. Some studies had investigated

381

that many microorganisms could reduce Fe(III) to Fe(II) using an assortment of electron

382

donors, such as acetate, lactate and H2. The most notable examples include: Geobacter

383

spp.49, Shewanella spp.50 and Geothrix fermentans51. Sulfur as the function of carbon

384

source might proceed Fe redox cycling in SICAD system. The discovery of Geothrix

385

has drawn attention in this study. Geothrix could release one or more electron-shuttling

386

compounds that promote Fe(III) oxide reduction and transfer electrons from the cell to

387

Fe(III) oxide52. The redox cycling of Fe electron shuttles secreted by Geothrix from

388

iron-oxide minerals may contribute to microbial cooperative catabolism. This indicated

389

that the iron cycle process might further promote the electrons transfer between two

390

solids (ie. Sulfur and iron(II) carbonate particles ) or two bacteria by electron shuttles.

391

In sum, the possible synergistic influence of SICAD on the enhanced nitrate

392

removal might be attributed to (1) the acid produced through sulfur driven

393

denitrification was found to promote the Fe(II) leaching from the ore and likely extend


Page 14 of 32

Page 15 of 32


394

the reaction zone from the surface to the liquid. As a result, more biomass was

395

accumulated in the SICAD system compared with the controls (sulfur, iron(II)

396

carbonate ore and SLAD systems). (2) circumneutral pH might be benefit for

397

denitrification bacteria activity (3) SICAD might bring out the redox cycling of Fe in

398

order to promote the transfer of electrons between two solids or two bacteria.

399

4.2 Low accumulation of N2O in SCIAD

400

In particular, the synergistic effect of SICAD not only improved nitrate removal

401

but also decreased NO2--N accumulation and the N2O release. A relationship exists

402

between N2O release, nitrite accumulation and pH was shown in Table 2. NO2--N and

403

N2O accumulation was likely caused by that nitrite reduction rate was lower than nitrate

404

reduction rate and N2O reduction rate was lower than nitrite reduction rate. The

405

synergistic effect of SICAD might improve the denitrification rate (nitrate reduction,

406

nitrite reduction and N2O reduction) compared with other systems. Hence, it led to

407

lowest NO2--N and N2O accumulation in SICAD system. Previous studies had shown

408

that both nitrite and pH also influence N2O reduction. Low pH was suggested to be an

409

important influential factor leading to N2O accumulation as an inter-mediate during

410

denitrification53. Hence, the N2O accumulation was highest in sulfur system. In addition,

411

FNA which was jointly determined by nitrite and pH, was likely a strong inhibitor of

412

N2O reduction54,55. At different HRTs, N2O accumulation increased with the decrease

413

of HRTs from 1.5 h to 0.5 h. The pH from 6.70±0.10 to 6.32±0.2, and the highest value

414

was 0.784 mg/L in SAD system. Then with the decrease of HRT form 1.5 h to 0.5 h,

415

the N2O production decreased to 0.12 mg/L. The tendency of the N2O concentration

416

with increment firstly was because the N2O accumulation was limited by the incomplete

417

of denitrification with the decreasing of HRT. The decreases of the nitrite reduction

418

rates at pH of 6.32 (HRT of 1.5 h) and 6.20 (HRT of 0.5 h) were likely due to FNA

419

inhibitions. At pH 6.32 and 6.20, this would give rise to FNA concentrations of 0.004

420

and 0.01 mg HO2-N/L at room temperature. These concentrations were in the range of

421

FNA that had previously been found to inhibit nitrite reduction56. Zhou et al.56 showed

422

that nitrite reduction by a EBPR sludge was inhibited by 40% at an FNA concentration

423

of 0.01 mg HO2-N/L. Compared with SAD system, FNA concentration and NO2-



424

production were lower in SLAD system leading to less N2O accumulation. In ICAD

425

system, the lower nitrate reduction rate might result in the lower NO2- production and

426

N2O accumulation.

427

4.3 Advantages of SICAD in environmental application

428

SICAD decreased sulfate release compared with SAD and SLAD systems, which

429

would reduce the risk of exceeding sulfate discharge limit. In all sulfur based

430

denitrification processes (ie. Sulfur or SLAD reactor), sulfate formation leads to high

431

sulfate content in treated effluent57. It should be controlled in case of meeting certain

432

standards. The allowable limit of sulfate for drinking water was set of 250 mg/L.

433

Theoretically, around 33 mg/L NO3--N could be denitrified without exceeding the

434

above limit. If there was some background value of sulfate in the water, the control of

435

excess sulfate production is a serious challenge. According to the sulfate formation per

436

nitrate removal (Figure 3 (A)), approximately 33.24, 36.92 and 32.74 mg/L NO3--N

437

could be denitrified in SAD, SICAD and SLAD system, respectively. Combining

438

autotrophic and heterotrophic denitrification is an effective strategy to control sulfate

439

formation. Hence, the 3.68 mg/L (=36.92-33.24) NO3--N could be denitrified by 9.63

440

mg/L methanol as additional carbon sources. It would need an accurate dosing of

441

methanol. If not, it would result in the secondary pollution due to the COD exceeding

442

the limit. With the decrease of HRT, the contribution for denitrification of iron(II)

443

carbonate increased from 8% to 42% and the contribution of sulfur decreased from 92%

444

to 58% in SICAD system. It suggested that at shorter HRTs, sulfur played little role in

445

denitrification in SICAD system, which would be suitable for practical application as a

446

highly efficient post-denitrification process.

447

In this study, the synergistic effect of SICAD could improve nitrate reduction,

448

decrease N2O release and produce less sulfate products. The study of autotrophic

449

denitrification in regard to coupling sulfur and iron(II) carbonate has received little

450

attention. This study revealed both the feasibility and mechanism of SICAD, offering

451

greater potential and feasibility for practical applications for secondary effluent

452

purification. However, several important research gaps and challenges exist for further

453

advancing sulfur-iron(II) carbonate-based cleanup technology. The presence of ferric


Page 16 of 32

Page 17 of 32


454

hydroxide as a product of the NAFO process indicated that phosphorus can be removed

455

by adsorption. Studying the synergistic effect of SICAD may also be important to gain

456

further insight into simultaneous phosphorus and nitrogen removal. However, the ferric

457

hydroxide wrapped on the surface of iron(II) carbonate may influence ferrous

458

contacting with contaminants, which should be further investigated.

459

Acknowledgments

460

The research was supported by the National Science Foundation of China

461

(No.21707156 and No. No. 51878652) and Funding for the 63th China postdoctoral

462

Science Foundation(No.Y8H1C91704).

463

References

464 465

(1) Van Rijn, J.; Tal, Y.; Schreier, H.J. Denitrification in recirculating systems: theory and applications. Aquac. Eng. 2006, 34, 364-376.

466

(2) Kim, I.S.; Oh, S.E.; Bum, M.S.; Lee, J.L.; Lee, S.T. Monitoring the denitrification of

467

wastewater containing high concentrations of nitrate with methanol in a sulphur-packed reactor.

468

Appl. Microbiol. Biotechnol. 2002, 59, 91-96.

469

(3) Wang, J.; Lu, H.; Chen, G.H.; Lau, G.N.; Tsang, W.L.; Van Loosdrecht, M.C.M. A novel

470

sulfate reduction, autotrophic denitrification, nitrification integrated (SANI) process for saline

471

wastewater treatment. Water Res. 2009, 43, 2363-2372.

472 473 474 475 476 477 478 479

(4) Di Capua, F.; Papirio, S.; Lens, P.N.L.; Esposito, G. Chemolithotrophic denitrification in biofilm reactors. Chem. Eng. J. 2015, 280, 643-657. (5) Zeng, H.; Zhang, T.C. Evaluation of kinetic parameters of a sulfur-limestone autotrophic denitrification biofilm process. Water Res. 2005, 39: 4941-4952. (6) Weber, K.A.; Achenbach, L.A.; Coates, J.D. Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 2006, 4 (10), 752-764. (7) Chaudhuri, S.K.; Lack, J.G.; Coates, J.D. Biogenic magnetite formation through anaerobic biooxidation of Fe(II). Appl. Environ. Microbiol. 2001, 67, 2844-2848.

480

(8) Clément, J.C.; Shrestha, J.; Ehrenfeld, J.G.; Jaffé, P.R. Ammonium oxidation coupled to

481

dissimilatory reduction of iron under anaerobic conditions in wetland soils. Soil Biol. Biochem.

482

2005, 37, 2323-2328.

483 484

(9) Laube, N.; Frimmel, H.; Hoernes, S.; Oxygen and carbon isotopic study on the genesis of the Steirischer Erzberg iron(II) carbonate deposit (Austria). Miner. Deposita. 1995, 30, 285-293.

485

(10) Kuznetsov, A.; Krupenin, M.; Ovchinnikova, G..; Gorokhov, I.; Maslov, A.; Kaurova, O.;

486

Ellmies, R. Diagenesis of carbonate and iron(II) carbonate deposits of the Lower Riphean Bakal



487

Formation, the southern Urals: Sr isotopic characteristics and Pb-Pb age. Lithol. Miner. Resour.

488

2005, 40, 195-215.

489 490

(11) Shen, B.; Liu, F.Y.; Zhu, Z.M. Mechanism of stratabound iron(II) carbonate deposit in center Yunnan. Miner. Resour. Geol. 2006, 3, 011.

491

(12) Zhao, L.; Dong, H.; Kukkadapu, R.; Agrawal, A.; Liu, D., Zhang, J.; & Edelmann, R.

492

E. Biological oxidation of Fe(II) in reduced nontronite coupled with nitrate reduction by

493

Pseudogulbenkiania sp. strain 2002. Geochimica et Cosmochimica Acta. 2013, 119, 231-247.

494

(13) Yang, Y.; Chen, T.; Zhang, X.; Qing, C.; Wang, J. Simultaneous removal of nitrate and

495

phosphate from wastewater by siderite based autotrophic denitrification. Chemosphere. 2018, 199,

496

130-137.

497

(14) P, Scherer.; H, Lippert.; G, Wolff. Composition of the major elements and trace elements

498

of 10 methanogenic bacteria determined by inductively coupled plasma emission spectrometry.

499

Biological Trace Element Research. 1983, 5(3), 149-163.

500

(15) Cheng, H. Y.; Tian, X. D.; Li, C. H.; Wang, S. S.; Wang, A. J. Microbial

501

Photoelectrotrophic Denitrification as a Sustainable and Efficient Way for Reducing Nitrate to

502

Nitrogen. Environmental Science & Technology. 2017, 51, 12948-12955.

503

(16) APHA, 2005. Standard Methods for the Examination of Water and Wastewater.

504

(17) A, C. Anthonisen.; R, C. Loehr.; T, B.S. Prakasam.; E, G. Srinath. Inhibition of

505

nitrification by ammonia and nitrous acid. Journal WPCF. 1976, 48(5), 835-852.

506

(18) Peretyazhko, T.S.; Zachara, J.M.; Kukkadapu, R.K.; Heald, S.M.; Kutnyakov, I.V.; Moore,

507

D.A . Pertechnetate (TcO4-) reduction by reactive ferrous iron forms in naturally anoxic, redox

508

transition zone sediments from the Hanford Site, USA. Geochimica et Cosmochimica Acta. 2012,

509

92, 48-56.

510

(19) Morris, R. V.; Klingelhoefer, G.; Bernhardt, B., Schröder, C.; Rodionov, D. S.; De Souza,

511

P. A. & Kankeleit, E. Mineralogy at Gusev Crater from the Mössbauer spectrometer on the Spirit

512

Rover. Science. 2004, 305(5685), 833-836.

513

(20) Wang, A.; Cheng, H.; Ren, N.; Cui, D.; Lin, N.; & Wu, W. Sediment microbial fuel cell

514

with floating biocathode for organic removal and energy recovery. Frontiers of Environmental

515

Science & Engineering. 2012, 6(4), 569-574.

516 517

(21) Zhou, J.Z.; Bruns, M.A.; Tiedje, J.M. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 1996, 62 (2), 316-322.

518

(22) Caporaso, J.G.; Lauber, C.L.; Walters, W.A.; Berg-Lyons, D.; Huntley, J.; Ultra-high-

519

throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. Isme J. 2012,

520

6 (8), 1621-1624.

521

(23) Hammes, F.; Goldschmidt, F.; Vital, M.; Wang, Y. Y. & Egli, T. Measurement and


Page 18 of 32

Page 19 of 32


522

interpretation of microbial adenosine tri-phosphate (ATP) in aquatic environments. Water Res. 2010,

523

44, 3915-3923.

524

(24) Zhang, Z. J.; Qu, Y.Y.; Li, S.Z.; Feng, K. Soil bacterial quantification. approaches

525

coupling with relative abundances reflecting the changes of taxa. Scientific Reports. 2017, 4837(7),

526

1-11.

527

(25) Frederik, Hammes.; Felix, Goldschmidt.; Marius, Vital. Measurement and interpretation

528

of microbial adenosine tri-phosphate (ATP) in aquatic environments. Water Res. 2010, 44, 3915-

529

3923.

530

(26) Zhou, A.; Guo, Z.; Yang, C.; Kong, F.; Liu, W.; Wang, A. Volatile fatty acids productivity

531

by anaerobic co-digesting waste activated sludge and corn straw: effect of feedstock proportion. J.

532

Biotechnol. 2013, 168(2), 234-239.

533

(27) Y, Sun.; M, Nemati. Evaluation of sulfur-based autotrophic denitrification and

534

denitritation for biological removal of nitrate and nitrite from contaminated waters. Bioresource

535

Technology. 2012,114,207-216.

536

(28) Bishop, J. L.; Quinn, R. & Dyar, M. D. What Lurks in the Martian Rocks and Soil?

537

Investigations of Sulfates, Phosphates, and Perchlorates Mössbauer parameters of iron in phosphate

538

minerals: Implications for interpretation of martian data. American Mineralogist. 2014, 99, 914-942.

539

(29) Holm-Hansen, O. Determination of microbial biomass in ocean profiles. Limnol.

540

Oceanogr. 1969, 14, 740-747

541

(30) Velten, S.; Hammes, F.; Boller, M.; Egli, T. Rapid and direct estimation of active biomass

542

on granular-activated carbon through adenosine tri-phosphate (ATP) determination. Water Res,

543

2007, 41, 1973-1983.

544

(31) Freguia, S.; Rabaey, K.; Yuan, Z. & Keller, J. Electron and carbon balances in microbial

545

fuel cells reveal temporary bacterial storage behavior during electricity generation. Environmental

546

Science & Technology. 2007, 41(8), 2915-2921.

547

(32) Schmalenberger, A.; Hodge, S.; Bryant, A.; Hawkesford, M.J.; Singh, B.K. & Kertesz,

548

M.A. The role of Variovorax and other Comamonadaceae in sulfur transformations by microbial

549

wheat rhizosphere communities exposed to different sulfur fertilization regimes. Environmental

550

Microbiology. 2008, 10(6), 1486-1500.

551 552

(33) Zhang, T. C.; Lampe, D. G. Sulfur: limestone autotrophic denitrification processes for treatment of nitrate-contaminated water: batch experiments. Water Res. 1999, 33, 99-608.

553

(34) Sobolev, D. & Roden, E. E. Suboxic deposition of ferric iron by bacteria in opposing

554

gradients of Fe(II) and oxygen at circumneutral pH. Appl Environ Microbiol. 2001, 67,1328-1334.

555

(35) Johanna, V. Weiss.; Jeremy, A. Rentz.; Todd, Plaia. Characterization of Neutrophilic

556

Fe(II)-Oxidizing Bacteria Isolated from the Rhizosphere of Wetland Plants and Description of



557

Ferritrophicum radicicola gen. nov. sp. nov., and Sideroxydans paludicola sp. nov.

558

Geomicrobiology Journal. 2007, 24,559-570.

559

(36) Hafenbradl, D.; Keller, M.; Dirmeier, R.; Rachel, R.; Robanagel, P.; Burggraf, S.; Huber,

560

H.; Stetter, K.O. Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that

561

oxidizes Fe2+ at neutral pH under anoxic conditions.pdf. Arch. Microbiol. 1996, 166, 308-314.

562

(37) Dong, J.W.; Yong, D.L.; Yi, Y.W.; Hong, J.W. Simultaneous bio-autotrophic reduction

563

of perchlorate and nitrate in a sulfur packed bed reactor: Kinetics and bacterial community structure.

564

Water Res. 2017, 108, 280-292.

565

(38) Kelly, P. Nevin.; Derek, R. Lovley. Mechanisms for Accessing Insoluble Fe(III) Oxide

566

during Dissimilatory Fe(III) Reduction by Geothrix fermentans. Applied and Environmental

567

Microbiology. 2002, 5, 2294-2299.

568 569 570 571

(39) Thompson, A.; Chadwick, O. A.; Rancourt, D. G.; Chorover, J. Iron-oxide crystallinity increases during soil redox oscillations. Geochim. Cosmochim. Acta .2006, 70 (7), 1710-1727. (40) Straub, K.L.; Benz, M.; Schink, B.; Widdel, F. Anaerobic, nitrate-dependent microbial oxidation of ferrous iron. Appl. Environ. Microbiol. 1996, 62 (4), 1458-1460.

572

(41) Weiss, J. V.; Rentz, J. A.; Plaia, T.; Neubauer, S. C.; Merrill-Floyd, M.; Lilburn, T.

573

Characterization of neutrophilic Fe(ii)-oxidizing bacteria isolated from the rhizosphere of wetland

574

plants and description of Ferritrophicum radicicola gen. nov. sp. Nov., and Sideroxydans paludicola

575

sp. Geomicrobiol. J. 2007, 24, 559-570.

576

(42) Fleming, E. J.; Davis, R. E.; McAllister, S. M.; Chan, C. S.; Moyer, C. L. Hidden in plain

577

sight: discovery of sheath-forming, iron-oxidizing Zetaproteobacteria at Loihi Seamount, Hawaii,

578

USA. FEMS Microbiology Ecology. 2013, 85(1), 116-127.

579 580

(43) Rakshit, S.; C, J. Matocha.; M, S. Coyne. Nitrite reduction by siderite. Soil chemistry. 2008. 72:1070-1077.

581

(44) Straub, K. L.; Schönhuber, W. A.; Buchholz-Cleven, B. E. & Schink, B. Diversity of

582

ferrous iron-oxidizing, nitrate-reducing bacteria and their involvement in oxygen-independent iron

583

cycling. Geomicrobiol J. 2004, 21, 371-378.

584 585

(45) Yue, wang.; Charles, Bott.; Robert, Nerenberg. Sulfur-based denitrification: Effect of biofilm development on denitrification fluxes. Water Res. 2016, 100, 184-193.

586

(46) Kelly, D. P.; & Wood, A. P. Confirmation of Thiobacillus denitrificans as a species of the

587

genus Thiobacillus, in the bsubclass of the Proteobacteria,a with strain NCIMB 9548 as the type

588

strain. International Journal of Systematic and Evolutionary Microbiology. 2000, 50, 547-550.

589

(47) Theodore, M. Flynn.; Edward, J. O’Loughlin.; Bhoopesh, Mishra.; Thomas, J.

590

DiChristina.; Kenneth, M. Kemner. Sulfur-mediated electron shuttling during bacterial iron

591

reduction. Science. 2014, 344, 6187.


Page 20 of 32

Page 21 of 32


592 593

(48) Sabrina, Hedrich.; Michael, Schlomann.; D, Barrie. Johnson. The iron-oxidizing proteobacteria. Microbiology. 2011, 157, 1551-1564.

594

(49) Lovley, D. R. & Phillips, E. J. P. Novel mode of microbial energy metabolism: organic

595

carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol.

596

1988, 54, 1472-1480.

597 598

(50) Myers, C. R.; & Nealson, K. H. Microbial reduction of manganese oxides: interactions with iron and sulfur. Geochim. Cosmochim. Acta .1988,52, 2727–2732.

599

(51) Coates, J. D.; Ellis, D. J.; Gaw, C. V.; & Lovley, D. R. Geothrix fermentans gen.nov., sp.

600

Nov., a novel Fe(iii)- reducing bacterium from a hydrocarbon-contaminated aquifer. Int. J. Syst.

601

Bacteriol. 1999, 49, 1615–1622.

602 603

(52) Lovley, D. R.; Holmes, D. E.; & Nevin, K. P. Dissimilatory Fe(III) and Mn(IV) reduction. Adv Microb Physiol. 2004, 49: 219-286.

604 605

(53) Kampschreur, M.J.; Temmink, H.; Kleerebezem, R.; Jetten, M.S.M.; van Loosdrecht, M.C.M. Nitrous oxide emission during wastewater treatment. Water Res. 2009, 43, 4093-4103.

606 607

(54) Itokawa, H.; Hanaki, K.; Matsuo, T. Nitrous oxide production in high-loading biological nitrogen removal process under low COD/N ratio condition. Water Res. 2001, 35 (3), 657−664.

608

(55) Chen, P.; DeBeer George, S.; Cabrito, I.; Antholine, W. E.; Moura, J. J.; Moura, I. &

609

Solomon, E. I. Electronic Structure Description of the μ4-Sulfide Bridged Tetranuclear CuZ Center

610

in N2O Reductase. JACS. 2002, 124(5), 744-745.

611

(56) Zhou, Y.; Pi, J.M.; Yuan, Z. G. Free nitrous acid inhibition on anoxic phosphorus uptake

612

and denitrification by poly-phosphate accumulating organisms. Biotechnol. Bioeng. 2007, 98, 903-

613

912.

614

(57) Sahinkaya, E.; Kilic, A. & Duygulu, B. Pilot and full scale applications of sulfur-based

615

autotrophic denitrification process for nitrate removal from activated sludge process effluent. Water

616

Res. 2014, 60, 210-217.

617 618

Figure Captions

619

Figure 1 Nitrate removal performances of four systems (A) and TN removal performances of four

620

systems (B) with different electron donors (SAD, ICAD, SICAD and SLAD) at different HRTs

621

Figure 2 Nitrogen balance of nitrogen compounds (left axis) with different electron donors

622

(SAD (A), ICAD (B), SICAD (C) and SLAD (D)) at different HRTs (green is nitrogen, yellow is

623

N2O-N, blue is NO2--N and slash is NO3--N) The right axis is N2O production (mg/L)

624

Figure 3 The sulfate production during per nitrate removal (A), Ferrous leaching (B) and

625

Mössbauer spectra of substances on the iron(II) carbonate particles in SICAD system



626

at 77 K (C).

627

Figure 4 The classification of 16s rRNA gene sequences from the microbial

628

communities on the filter surfaces in the four reactors at the phylum level (A). The

629

microbial communities on the filter surfaces in the four reactors at the genus level

630

(relative abundance ≥1%) (B). Heatmap (C) and PCoA (D) analyses of the microbial

631

communities

632

Figure 5 Possible metabolic path of sulfur coupled with iron(II) carbonate-driven

633

autotrophic denitrification between microorganisms

634

Table Captions

635

Table 1 pH,

636

donors at different HRTs

637

Table 2 The bacterial cell numbers derived from the ATP and protein measurements

638

Supporting Information (SI)

639

Figure S1 Natural iron(II) carbonate XRD patterns.

640

Figure S2 SEM images of biofilms on the surface of the filter in SAD system (a and b);

641

ICAD system (c and d); SICAD system (e and f); and SLAD system (g and h). (Left:

642

magnified 6000 times. Right: magnified 30000 times.)

643

Figure S3 Biomass production on the particles during per TN removal.

644

Table S1 The contributions of sulfur and iron(II) carbonate to the enhanced nitrate

645

removal in SICAD system

646

Table S2 List of some previous studies on S-based denitrification and nitrate-dependent

647

anaerobic oxidation processes to remove TN

NO3--N

removal, FNA, NO2- and N2O accumulation with different electron

648


Page 22 of 32

Page 23 of 32


649

650

651 652 653

Figure 1 Nitrate removal performances of four systems (A) and TN removal performances of four

654

systems (B) with different electron donors (SAD, ICAD, SICAD and SLAD) at different HRTs

655 656



Page 24 of 32

657

Table 1 pH, NO3--N removal ,FNA, NO2- and N2O accumulation with different electron

658

donors at different HRTs HRT(h)

Reactors

12

6

3

1.5

0.5

6.70±0.10

6.50±0.20

6.45±0.1

6.32±0.2

6.20±0.1

18.26

18.30

12.41

9.31

6.63

FNA

0

123×10-5

188×10-5

397×10-5

910×10-5

NO2--N

0

0.57

0.77

1.21

2.97

N2O

0.002

0.235

0.551

0.784

0.120

pH

7.80±0.2

7.67±0.2

7.62±0.1

7.41±0.3

7.40±0.2

14.46

8.16

4.38

3.44

2.17

0

4.2×10-5

7.8×10-5

20.3×10-5

22.3×10-5

pH --N

NO3 Sulfur

iron(II) carbonate

--N

NO3

FNA NO2--N

0

0.28

0.47

0.76

0.82

N2O

0.005

0.006

0.007

0.015

0.003

pH

7.24±0.1

7.11±0.1

7.01±0.1

6.82±0.2

6.80±0.1

--N

NO3 SICAD

SLAD

19.48

19.45

18.52

16.44

15.42

FNA

0

3.6×10-5

4.9×10-5

1.2×10-5

44.5×10-5

NO2--N

0

0.06

0.07

0.01

0.41

N2O

0.001

0.001

0

0

0.006

pH

7.60±0.3

7.55±0.2

7.39±0.2

7.3±0.1

7.23±0.2

NO3--N

19.02

18.29

14.87

14.12

11.22

FNA

0

0

12.8×10-5

3.91×10-5

57.2×10-5

0

0

0.46

0.11

1.41

0.003

0.001

0.010

0.014

0.030

--N

NO2

N2O

659 660


Page 25 of 32


662

663 664

Figure 2 Nitrogen balance of nitrogen compounds (left axis) with different electron donors

665

(SAD (A), ICAD (B), SICAD (C) and SLAD (D)) at different HRTs (green is nitrogen, yellow is

666

N2O-N, blue is NO2--N and slash is NO3--N) The right axis is N2O production (mg/L)

667 668



669

670

671 672

Figure 3 The sulfate production during per nitrate removal (A), Ferrous leaching (B)

673

and Mössbauer spectra of substances on the iron(II) carbonate particles in SICAD


Page 26 of 32

Page 27 of 32

674


system at 77 K (C).

675



676

Page 28 of 32

Table 2 The bacterial cell numbers derived from the ATP and protein measurements iron(II) carbonate

Sulfur in SICAD

0.78×108

0.04×108

7.11×108

±0.02×108

±0.13×107

0.12±0.03

0.025±0.008

Sulfur

ATP cell/mm2 Protein mg/mm2

iron(II) carbonate

Sulfur in SLAD

Limestone in SLAD

0.52×108

1.74×108

0.25×108

±0.37×108

±0.14×108

±0.56×108

±0.07×108

0.425±0.05

0.476±0.19

0.263±0.23

0.132±0.05

in SICAD

677


Page 29 of 32


679 680

681

682 683 684 685 686



687 688

689

690 691 692 693 694 695 696 697 698

Figure 4 The classification of 16s rRNA gene sequences from the microbial communities on the filter surfaces in the four reactors at the phylum level (A). The microbial communities on the filter surfaces in the four reactors at the genus level (relative abundance ≥ 1%) (B). Heatmap (C) and PCoA (D) analyses of the microbial communities


Page 30 of 32

Page 31 of 32


699 700

701 702 703

Figure 5 Possible metabolic path of sulfur coupled with iron(II) carbonate-driven

704

autotrophic denitrification between microorganisms

705



190x135mm (96 x 96 DPI)


Page 32 of 32

Coupled sulfur and iron(II) carbonate-driven autotrophic denitrification

Recommend Documents