Stratification of extracellular polymeric substances (EPS) for

Feb 27, 2017 - ABSTRACT: Sludge aggregation and biofilm formation are the most effective approaches to solve the washout of anammox microorganisms. In...
0 downloads 6 Views 1MB Size
Subscriber access provided by UNIVERSITY OF SOUTH CAROLINA LIBRARIES

Article

Stratification of extracellular polymeric substances (EPS) for aggregated anammox microorganisms Fangxu Jia, Qing Yang, Xiuhong Liu, Xiyao Li, Baikun Li, Liang Zhang, and Yongzhen Peng Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05761 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 27, 2017

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 free 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 accessible to all readers and 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.

Environmental Science & Technology 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 30

Environmental Science & Technology

1

Stratification of extracellular polymeric substances (EPS) for

2

aggregated anammox microorganisms

3

Fangxu Jia 1, Qing Yang 1,*, Xiuhong Liu 2, Xiyao Li 1, Baikun Li 1, Liang Zhang 1, Yongzhen Peng

4

1,*

5 6

1

7

Technology, Engineering Research Center of Beijing ,Key Laboratory of Beijing for Water Quality

8

Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing

9

100124, PR China

National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse

10

2

11

China

12

*Corresponding Author.

13

School of Environment & Natural Resources, Renmin University of China, Beijing 100872, PR

E-mail: [email protected] Tel: 86-10-67392627 Fax: 86-10-67392627

E-mail: [email protected] Tel: 86-10-67392627 Fax: 86-10-67392627

1

ACS Paragon Plus Environment

Environmental Science & Technology

14

ABSTRACT: Sludge aggregation and biofilm formation are the most effective approaches to

15

solve the washout of anammox microorganisms. In this study, the structure and composition of

16

EPS (extracellular polymeric substances) were investigated to elucidate the factors for the

17

anammox aggregation property. Anammox sludge taken from 18 lab-scale and pilot-scale

18

reactors treating different types of wastewater was analyzed using EEM-PARAFAC (excitation-

19

emission matrix and parallel factor analysis), FTIR (fourier transform infrared spectroscopy)

20

and real-time PCR combined with multivariate statistical analysis. The results showed that

21

slime and TB-EPS (tightly bound EPS) were closely related with water quality and sludge

22

morphology, and could be used as the indicators for anammox microbial survival ability and

23

microbial aggregate morphology. Furthermore, slime secreted from anammox bacterial cells

24

may be exhibited higher viscosity to the sludge surface and easily formed the gel network to

25

aggregate. Large amounts of hydrophobic groups of protein in TB-EPS promoted the microbial

26

aggregation. The mechanisms of anammox aggregation explored in this study enhanced the

27

understanding of anammox stability in wastewater treatment processes.

28 29

Keywords: Anammox, Extracellular polymeric substances (EPS), Aggregation behavior, Uronic

30

acids, β-sheet.

2

ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

31

Environmental Science & Technology

Table of Contents

32

3

ACS Paragon Plus Environment

Environmental Science & Technology

33

Page 4 of 30

Introduction

34

Application of anammox in wastewater treatment plants has been extensively studied, due to

35

its high efficiency, no need of oxygen and additional carbon source and low sludge output 1.

36

However, the anammox bacteria have slow growth rate with doubling time of two weeks 2, which

37

hinders the adoption of this technology. Maintaining high biomass concentrations and preventing

38

the loss of anammox biomass is critical for this process. To date, the most feasible and effective

39

method to improve the bacterial accommodation capacity are granulation, biofilm formation and

40

immobilized biomass 3.

41

EPS (extracellular polymeric substances) plays a crucial role in sludge granulation and biofilm

42

formation as well as the maintenance of the structural integrity 4. Their physicochemical properties

43

and spatial distribution structure can dramatically affect the structure and function of microbial

44

aggregate. For example, proteins, humic acids and uronic acids in EPS contributed to the

45

hydrophobicity of activated sludge while carbohydrates contributed to the hydrophilic nature 5. The

46

β-polysaccharides forms the outer layer to support the mechanical stability of aerobic granules 6.

47

Certain protein secondary structures promoted bioflocculation aggregation, adsorption and biofilm

48

formation 7, 8. Although EPS in activated sludge and biofilms have been extensively studied, the key

49

mechanism of aggregation ability of anammox microorganisms remains unknown. Since the unique

50

properties of anammox sludge including density dependent 9, high aggregation ability

51

aggregation morphology of broccoli shape 10, strong tendency to form granules 11, 12 and the ability

52

to secrete large amounts of EPS 13, the EPS of anammox sludge is different from that in conventional

53

activated sludge. Therefore, a new approach to elucidate EPS production in anammox sludge should

54

be explored. 4

ACS Paragon Plus Environment

10,

Page 5 of 30

55

Environmental Science & Technology

To date, only a few studies have focused on EPS in anammox-dominated mixed culture, and 13, 14.

56

mainly limited to the content of protein and polysaccharides using colorimetric method

57

Meanwhile, many factors including different treatment processes, operational conditions and water

58

quality

59

previous studies only compared one anammox sludge sample with that of nitrifying sludge or

60

denitrifying sludge sample

61

anammox cannot suitable for other anammox processes. In addition, EPS had different stratified

62

structure in sludge and each fraction possesses different physicochemical characteristics of organic

63

matters16, such as slime layers markedly impeded the sludge dewaterability16, LB-EPS (loosely

64

bound EPS) might inhibit bioflocculation, sedimentation and sludge dewatering17, and cause the

65

irreversible fouling in membrane bioreactors18. On the other hand, TB-EPS (tightly bound EPS) had

66

the gel-like characteristics and strong elasticity and might benefit flocculation16, 19, 20. Furthermore,

67

since anammox bacteria have not been cultivated as a pure culture 9, it is so significant to determine

68

the origin of EPS from different microbes in anammox-dominated mixed culture. Therefore, to

69

elucidate the effect of EPS on anammox microbial aggregation, the structure and composition of

70

stratified EPS should be investigated at the microscopic level.

15

influenced the properties of EPS (fractions, structure, composition, etc.), whereas the

7, 10.

Therefore, these obtained results related to the aggregation of

71

The objective of the study was to elucidate the relationship between the properties of stratified

72

EPS (fractions, structure, composition, etc.) and the properties of anammox biomass (macro-

73

structure, activity, microbial composition, etc.) by studying a variety of anammox-dominated

74

biomass samples collected from lab-scale and pilot-scale systems using multivariate statistical

75

analysis (principal component analysis and Pearson correlation analysis). This study devote to

76

explore the critical factors for anammox microbes aggregation, which provide a reference to 5

ACS Paragon Plus Environment

Environmental Science & Technology

77

enhance the understanding of anammox stability in wastewater treatment processes.

78 79

Materials and methods

80

Sludge samples

81

Anammox sludge samples were collected from 18 different lab-scale and pilot-scale reactors,

82

which treated wastewater from synthetic wastewater, domestic sewage, landfill leachate and reject

83

water and possess different sludge morphology (flocs, biofilm and granules). The characteristics of

84

these sludge samples were shown in Supplementary Table S1. The collected samples were stored at

85

4℃ immediately after sampling for further analysis.

86 87

Bacterial activity test

88

To elucidate the activities of ammonium oxidizing bacteria (AOB), nitrite oxidizing bacteria

89

(NOB) and anammox bacteria, a series of batch experiments were carried out. The sludge samples

90

were washed three times by oxygen-free water to avoid the oxygen inhibition, and then injected into

91

a 500 ml flask to measure AOB, NOB and anammox activity. The initial pH was 7.5 and temperature

92

was maintained at 35 ±1 ℃. Initial concentration of ammonia and nitrite was 30 mg/L for anammox

93

bacteria. As for AOB and NOB, ammonia and nitrite was respectively added to 30 mg/L and the

94

average DO was maintained at 3.0 ±0.5 mg/L.

95 96

EPS extraction protocol

97

The slime and LB-EPS extraction protocol was modified according to the research of Yu et

98

al.16. In brief, 40 ml of sludge sample was placed in a 50 ml centrifuge tube and centrifuged at 2000 6

ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30

Environmental Science & Technology

99

g for 15 min, then the bulk solution was collected as slime. The bottom sediments were collected

100

and resuspended to their original volumes with PBS. After that, the suspensions were centrifuged at

101

5000 g for 15 min and the bulk solution was collected as LB-EPS. The TB-EPS extraction protocol

102

was according to our previous work

103

resuspended to original volumes with PBS. The ultrasonic method was employed to extract the TB-

104

EPS from resuspended sediments at 20 kHz and 5.59 W/ml for 1 min. The extracted solutions were

105

centrifuged at 20,000g for 20 min. Organic matter in the bulk solution was comprised of the TB-

106

EPS. Polytetrafluoroethylene membranes with a pore size of 0.45 μm were used to remove the

107

particulates present in the slime, LB-EPS and TB-EPS solutions.

21.

Briefly, the bottom sediments were collected and

108 109

EEM fluorescence spectroscopy and PARAFAC analysis

110

To avoid any inner-filtering effects, the EPS sample was diluted with deionized water to ensure

111

that their absorbance at 200 nm was lower than 0.05 22. EEM fluorescence spectroscopy measure

112

method was according to our previous work 21. The fluorescence spectral parameters of fluorescence

113

index (FI) and humification index (HIX) value were calculated as the report of Gabor et al.23.

114

The PARAFAC is a three-way method that was used to model the EEM fluorescence data. The

115

principle of the PARAFAC could be found elsewhere 24, so there is no need to provide a detailed

116

description on it. The PARAFAC process was carried out in MATLAB 8.3 (MathWorks, Natick,

117

MA) with the DOMFluor Toolbox (http://www.models.life.ku.dk). To avoid the impact on the

118

component score caused by varying EPS concentrations in different samples, the EEMs were

119

normalized by dividing the spectra by the corresponding DOC concentrations

120

the Rayleigh and Raman scatters by Delaunay triangulation method and subtract the control Milli7

ACS Paragon Plus Environment

25

after removal of

Environmental Science & Technology

121

Q water, respectively. The outliers were examined by comparing the leverage. The number of

122

fluorescence components was determined by a validation method, including split half analysis,

123

residual analysis and visual inspection.

124 125

Quantitative real-time PCR Assay

126

Genomic DNA was extracted from about 0.2 g of freeze-dried sludge using the FastDNA SPIN

127

Kit for Soil (QBIOgene Inc.,Carlsbad, CA, USA), with a beating time of 30 s and a speed setting of

128

5.0. The concentration of the extracted DNA was determined with Nanodrop ND-1000 ultraviolet-

129

visible spectrophotometry (Thermo, USA). Quantitative real-time PCR were performed in

130

MX3000P Real-Time PCR system (Stratagene, La Jolla, CA) with SYBR Premix Ex TaqTM

131

(TAKARA, Dalian, China) and the assays were carried out in a volume of 20 μL reaction mixtures,

132

including 10 μL of SYBR Premix Ex Taq, 0.4 μL of ROX Reference Dye, 0.3 μL of each primer

133

(10 μM), and 2μL of tenfold-diluted DNA template (1–10 ng). The program of protocol of each

134

consisted of the following steps: 3 min at 95 ℃, followed by 40 cycles of 30 s at 95 ℃, 30 s at

135

corresponding annealing temperature, and 45 s at 72 ℃. The standard curves for each bacteria gene

136

copies were constructed from a series of 10-fold dilutions of the plasmid DNAs. The results with

137

amplification efficiency in a range from 90%-110% and correlation coefficient above 0.9 were

138

employed. Primers and corresponding annealing temperature are listed in Supplementary Table S2.

139 140

Fourier transform infrared spectroscopy

141

The EPS samples were lyophilized and ground with the infrared grade KBr and molded into a

142

disc to characterize the major functional groups in the EPS using a FTIR spectrometer (IRPrestige8

ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30

Environmental Science & Technology

143

21, Shimadzu, Tokyo, Japan). IRsolution (v1.10) operating software was used to generate and

144

process the FTIR Spectra. The amide I region (1700-1600 cm-1) in the EPS was further analyzed to

145

extract information regarding protein secondary structures. Furthermore, the second-derivative

146

spectra and deconvolution spectra of amide I region were carried out to resolve the overlapped peaks

147

with the minimum residual using Peakfit software (version 4.12, Seasolve Software Inc.). Moreover,

148

a ratio of α-helix/(β-sheet + random coil) was used to describe the tightness degree of protein

149

structure (Supporting Information (SI) Table S5) 10.

150 151

Chemical analysis

152

Proteins (PN), humic acid (HA), polysaccharides (PS) and uronic acids (UA) were measured

153

according to the research of Badireddy et al.8. The DNA content was measured by the diphenylamine

154

colorimetric method

155

Vario TOC analyzer (Elementar Analysensysteme Hanau, Germany). The UV254 was measured with

156

a spectrophotometer UV-4802 (UNICO, Shanghai, China). Specific ultraviolet light absorbance

157

(SUVA) was calculated as UV254/DOC to characterize aromaticity 27. SCOD, TSS and VSS contents

158

of sludge were analyzed according to the Standard Methods 28. Ammonium, nitrite, nitrate were

159

analyzed using Lachat QuikChem8000 Flow Injection Analyzer (Lachat Instrument, Milwaukee,

160

USA). DO, pH and temperature were measured by oxygen, pH and temperature probes (WTW 340i,

161

WTW Company). All standards were purchased from Sigma-Aldrich (St. Louis, USA), and the

162

chemical analyses were carried out in duplicate using chemicals of analytical grade.

26.

The dissolve organic carbon (DOC) in the filtrate was analyzed using a

163 164

Data Analyses and Statistical Methods 9

ACS Paragon Plus Environment

Environmental Science & Technology

165

To display variation in samples of a statistical population, the boxplot was used for data

166

visualization (Fig 1a and e). The relationships between anammox sludge samples and EPS

167

components characteristics were analyzed using the principal component analysis (PCA) method in

168

CANOCO 5.0 software. Pearson correlation analysis was showed in heatmaps using the Euclidean

169

distances and hierarchical cluster analysis in the “pheatmap” package of R 3.1.0 software.

170 171

Results and discussion

172

Chemical characterization of EPS components

173

Variations on the contents of the three spatial scales EPS components were examined in 18

174

samples taken from different reactors (Fig 1a), with significant difference in water quality and

175

sludge morphology (the detailed data are presented in Fig.S1). Compare to slime (15.61±9.92%)

176

and LB-EPS (6.46±3.02%), TB-EP (77.93±10.3%) was the major component of total EPS, which

177

was similar to previous findings.

178

distributed substance in slime. The predominant substance in LB-EPS and TB-EPS was humic acid

179

and protein, respectively. The ratios of protein to polysaccharides, protein to DNA and protein to

180

humic acid were 2.10±1.07, 6.38±7.88 and 1.48±0.57, respectively. The SUVA of EPS was: TB-

181

EPS (0.62±0.12) > slime (0.55±0.41) > LB-EPS (0.39±0.31). Higher SUVA values indicate a higher

182

concentration of organic matters with carbon-carbon double bonds, such as lignin, humic acid, PAHs

183

(polycyclic aromatic hydrocarbon), PCBs (polychlorinated biphenyls), aromatic protein, etc 30. TB-

184

EPS should contain large amounts of aromatic protein and other two scales contained a lot of humic

185

acid in DOC.

29.

Polysaccharides and humic acid were the most widely

186 10

ACS Paragon Plus Environment

Page 10 of 30

Page 11 of 30

187

Environmental Science & Technology

Fig.1 here

188 189

EEM fluorescence spectra of three EPS fractions in different anammox samples was

190

investigated using PARAFAC model (Fig.1 b-d). The decomposed three components were identified

191

as the characteristic peaks at Ex/Em of (215, 275)/342 nm, (205, 240, 305)/398.5 nm and (225, 240,

192

335)/434.5 nm, which represented the protein-like substance (C1), heme-like substance (C2) and

193

humic-like substance (C3) fluorophores, respectively (peak recognition of EEM fluorescence

194

spectra are presented in Supporting Information (SI) Text 1). C1 was the most widely distributed

195

substance in all three spatial scales EPS (45.19±16.29%, 46.88±18.42%, 74.26±18.40%) (Fig. 1e

196

and the detailed data are presented in Fig.S1). Unlike the TB-EPS, there was approximately the

197

same magnitude of C1 to C3 in slime and LB-EPS. The fluorescence intensity scores showed the

198

relative abundance of EPS components in three spatial scales was inconsistent with the results of

199

colorimetric method, which was caused by extremely high sensitivity of fluorescence spectroscopy

200

than chromatic spectrum 31. In addition, FI values in the three spatial scales EPS varied in a range

201

of 1.59±0.11~1.72±0.13, with relatively moderate values (1.4~1.7), indicating that the DOC in the

202

EPS was originated from both microbial metabolism and waste water 32. Except for sample F6/L

203

with a strong humic content of landfill leachate (HIX>6), the HIX values in slime and LB-EPS

204

ranged from 1.37±0.59 to 1.57±0.73, representing the character of weak humic content and high

205

microbial metabolic activity 33 that was consistent with the high SUVA values in slime and LB-EPS.

206

Low levels of TB-EPS (HIX=0.49±0.16) illustrated the TB-EPS components were originated from

207

from bacterial secretion rather than exogenous source.

208 11

ACS Paragon Plus Environment

Environmental Science & Technology

209

Quantification of EPS components using PCA method

210

The compositional characteristics of EPS based on the chemical and spectral analysis was

211

correlated with the activities of AOB, NOB and anammox bacteria using PCA method. By

212

projecting the sample points onto the arrows of component concentration, samples were centered

213

satisfactorily on the basis of water quality (Fig.2 a-3). Synthetic wastewater samples (bottom-left)

214

were separated from domestic sewage (upper-right). The C1, C2 and PN had a relatively high

215

abundance in the domestic sewage, while PS, FI, DOC, UA and HIX were more abundant in

216

synthetic wastewater (Fig.2 a-1). In addition, landfill leachate and reject water samples were located

217

in the upper-left side (Fig.2 a-3), showing a strong humification depended upon high content of

218

SUVA, DNA, HA, UV254 and C3 (Fig.2 a-1). However, LB-EPS samples were scattered in PCA

219

scatterplot of both water quality and sludge morphology (Fig.2 b-2 and b-3), whereas there was a

220

clear clustering of the TB-EPS samples based on sludge morphology (Fig.2 c-2). Biofilm samples

221

clustered on the upper-middle side of the plot (Fig.2 c-2), which had high levels of UA, C1 and PS

222

(Fig.2 c-1), while granule samples located in the bottom-left (Fig.2 c-2) that were characterized by

223

high SUVA and UV254 richness (Fig.2 c-1). Furthermore, flocs samples were apart from other two

224

types of sludge and occupied a large area in the middle of the scatterplot (Fig.2 c-2), so that the

225

distribution of each component in flocs was comparatively uniform.

226

Fig.2 here

227 228

PCA results indicated that different clusters represented different types of water quality and

229

sludge morphology. The slime layer was correlated with water quality type due to its outermost

230

spatial scales location. This might be helpful for identifying microbial survival environment. The 12

ACS Paragon Plus Environment

Page 12 of 30

Page 13 of 30

Environmental Science & Technology

231

TB-EPS layer was associated with microbial aggregates morphology, since the gel-like property of

232

TB-EPS was tightly bounded to the cell surface and played an important role for flocculation

233

Bacterial activity (the Supporting Information Table S1) showed that nitrification activity of AOB

234

and NOB were higher in the flocs, whereas the anammox activity was higher in the granules (Fig.2

235

c-1), which was consistent with previous findings that nitrification mostly occurred in small flocs

236

while anammox was found in large aggregates 35. Since loose structure of the flocs have lower mass

237

transfer resistance, ammonium and oxygen was easily penetrated in to the flocs, which benefited

238

the growth of nitrifying bacteria (AOB and NOB). While large aggregates had high retention

239

capability to accommodate microorganisms with slow growth rates (e.g. anammox bacteria) and

240

provided anoxic micro-environment.

34.

241 242

Pearson correlation analysis of anammox sludge aggregation

243

Relationships of functional microorganisms and EPS parameters were established to explore

244

the aggregation behavior of anammox sludge using Pearson correlation analysis (Fig.3). The real-

245

time PCR and FTIR results showed many positive and negative correlations (Supporting

246

Information Text 2, Table S4 and S5). In this study, we only focused on the relationships associated

247

with anammox microorganisms. For slime layer, the abundance of anammox were positively

248

correlated with the level of UA and DOC but was negatively correlated with SUVA, indicating that

249

the more abundance of anammox in sludge, the more slime was secreted that was characterized by

250

high UA content and low carbon-carbon double bonds (Fig.3(a)). For TB-EPS layer, anammox

251

abundance was positively correlated with the protein secondary structures of β-sheet in TB-EPS

252

layer (Fig.3(c)). Based on these results, two relationships: anammox and UA in slime; anammox 13

ACS Paragon Plus Environment

Environmental Science & Technology

253

and β-sheet in TB-EPS were elaborated.

254 255

Fig.3 here

256 257

Table 1 here

258 259

For the first positive correlation between anammox and UA in slime (Fig.4 a-1), anammox

260

bacteria were able to synthesize UA. Given the metagenome of Candidatus Kuenenia stuttgartiensis,

261

one gene named kustb0219 was found to encoded GDP-mannose dehydrogenase synthesis (algD)

262

36,

263

the ability to synthesize UA. In addition, the FTIR spectra of slime demonstrated typical bands of

264

UA (Fig.4 a-2 and Table 1): the bands at 1655 cm-1 and 1402 cm-1 were corresponded to the

265

asymmetric and symmetric stretching vibration of COO- attributed to the presence of UA. The band

266

at 1540 cm-1 was assigned to the C=C in pyranose ring, which might be caused by the β-elimination

267

effect of alginate lyases. The band at 1240 cm-1 was assigned to the presence of O-acetyl ester for

268

bacterial alginates 38. The bands at 952 cm-1 showed a weak absorbance that might be caused by the

269

existence of nucleic acids 8. From a structural point of view, UA is the unique component in alginate,

270

which is an unbranched exopolysaccharide composed of random arranged 1, 4-linked uronic

271

residues of β-d-mannuronate (M) and α-l-guluronate (G) 39, and typically occurred as (–G–)n, (–

272

M–)n and (–MG–)n blocks (Fig.4c) 40. The (–G–)n blocks are a rod-like polymer yielding an array

273

of coordination sites that benefit divalent cations in their cavities and provide gel-forming capacity

274

(Fig.4c). In contrast, (–M–)n and (–MG–)n blocks are fiber-like chain necessary for the flexibility of

which was known as crucial enzymes for alginate biosynthesis 37. Hence, anammox bacteria have

14

ACS Paragon Plus Environment

Page 14 of 30

Page 15 of 30

Environmental Science & Technology

275

the chains, the connection of (–G–)n blocks and the network structure during gelation 38. Alginate

276

was involved in the development of microcolonies and responsible for the mechanical stability of

277

biofilms

278

affected by UA 5, 8. Since the viscosity of slime was increased with alginate concentration, UA in

279

the slime of anammox bacteria may be showed stronger viscosity, and plays a key role for the

280

adhesive ability and architecture structures of anammox sludge 38, 39, 41.

39.

In addition, bioflocculation, settling and dewatering properties were substantially

281

For the second positive correlation between anammox and β-sheet in TB-EPS (Fig.4 b-1),

282

secondary structure of protein in EPS played important roles in aggregation, adhesion,

283

bioflocculation and biofilm formation

284

bioflocculation 8, and β-sheet contributed to high aggregation ability of anammox sludge 10. Due to

285

the twisted and pleated sheet structure of β-sheet (Fig. 4c), large amounts of inner hydrophobic

286

groups of amino acids were more easily to be exposed and express the hydrophobic property of

287

anammox sludge 10. Increasing the hydrophobicity of cell surfaces promoted cell-to-cell aggregation

288

42,

289

spectra showed more hydrophobic functional groups in TB-EPS (Fig. 4 b-2 and Table 1): the peak

290

at approximately 1668 cm-1 and 1623 cm-1 were mainly assigned to C=C and C=O in proteins. The

291

band at 1384 cm-1 was attributed to the bending vibration of C-H in -CH3. There were fewer

292

hydrophilic functional groups (especially for high polarity of N-H, -OH and COO-) in TB-EPS.

293

Relative content of β-sheet in TB-EPS proteins increased with the abundance of anammox bacteria

294

(Fig. 4 b-1), implying that TB-EPS secreted from anammox bacteria had a strong hydrophobicity to

295

promote microbial aggregation.

7, 8.

Previous studies showed that β-sheets promoted

and the cell surface hydrophobicity was the triggering force for bio-granulation

296 15

ACS Paragon Plus Environment

43.

The FTIR

Environmental Science & Technology

297

Fig.4 here

298 299

Overall, the mechanisms of anammox aggregation might be contain three stages. (1) Initial

300

attachment of anammox cells to the surface: Free anammox bacteria swimming in close proximity

301

to abiotic solid surface until find an nutritious habitat for initial contact by secrete small amounts of

302

sticky UA in bulk water; (2) Production of UA resulting in more firmly irreversible adherence: Then

303

they come to rest on the surface and contact with other cells. In this moment, many anammox

304

bacteria began to secrete a lot of UA. The gelation properties of UA can play a role in net capturing

305

to prevent cells detachment due to its three dimensional network structure. (3) Development of

306

microcolony architecture: After that, during the growth and reproduction of anammox bacteria,

307

more EPS is produced and tightly-wrapped on cell surface. Due to the β-sheet secondary structure

308

in extracellular protein, inner hydrophobic groups were more easily to be exposed, which makes the

309

surface of anammox community showed higher hydrophobic property. It can promote cell-to-cell

310

aggregation and form strong microcolony architecture, which make them undergo further adaptation

311

to life in large aggregates.

312 313

Significance of this study

314

The identification of the key role of stratified EPS is of importance to understand the

315

mechanisms of anammox microbial aggregation and enhance anammox efficiency through biofilms

316

and granules formation. By examining anammox-dominated mixed culture samples from 18

317

anammox systems, this study for the first time elucidated the characteristics of slime and TB-EPS

318

composition and their correlation with water quality and sludge morphology. UA (in slime) and β16

ACS Paragon Plus Environment

Page 16 of 30

Page 17 of 30

Environmental Science & Technology

319

Sheet (in TB-EPS) level were positively correlated with anammox abundance (Fig. 4a). To improve

320

the accommodation capacity of anammox biomass, mature anammox sludge should be added in

321

order to promote the release of UA or directly added alginate to make the flocs quickly form granules

322

or biofilm. Another option will be adding accelerants (e.g. quorum sensing molecules

323

pretreatment (e.g. ultrasound

324

activity that may promote anammox bacterial secrete more UA. In addition, Verrier et al. found that

325

hydrophobic surfaces are favor adhesion of hydrophobic bacteria 49, Based on the above result, the

326

high hydrophobic characteristic of anammox can be utilized to develop the special carriers or small

327

particles (acting as crystal nucleus) with high hydrophobicity (eg. polytetrafluorethylene,

328

polypropylene or polyethylene 49) that can priority selection bond anammaox bacterial cells to form

329

biofilms or granules 50. Future studies should be extracted alginate-like exopolysaccharides from

330

anammox sludge and verified its gel networks by gel formation experiments and further elucidated

331

the role of stratified EPS on the anammox aggregation behavior by proteomics and glycomics.

47

and magnetic field

48)

45, 46)

or

to enhance anammox bacterial metabolic

332 333

ASSOCIATED CONTENT

334

Supporting Information

335

The Supporting Information is available free of charge on the ACS Publications website at DOI:

336

Additional discussion details Text1-Text2 and Table S1-S5 and Figures S1−S5. (PDF)

337 338

AUTHOR INFORMATION

339

Corresponding Author

340

*Tel: 86-10-67392627; E-mail: [email protected] (Yongzhen Peng) 17

ACS Paragon Plus Environment

Environmental Science & Technology

341

*Tel: 86-10-67392627; E-mail: [email protected] (Qing yang)

342

Notes

343

The authors declare no competing financial interest.

344 345

ACKNOWLEDGEMENTS

346

We greatly thank Dr. Shanyun Wang, Dr. Lei Miao, Dr. Xiaoxia Wang, Dr. Rui Du, Dr.Yandong

347

Yang, Mr. Pengchao Gu and Miss. Han Xiao for their kind help in sampling on this work. This

348

research was financially supported by Nature Science Foundation of China (21677005) and the

349

Funding Projects of Beijing Municipal Commission of Education.

350 351

REFERENCE

352

(1) Jetten, M. S. M.; Wagner, M.; Fuerst, J.; Loosdrecht, M. V.; Kuenen, G.; Strous, M., Microbiology

353

and application of the anaerobic ammonium oxidation ('anammox') process. Curr. Opin. Biotech. 2001,

354

12, (3), 283-8.

355

(2) Strous, M.; Pelletier, E.; Mangenot, S.; Rattei, T.; Lehner, A.; Taylor, M. W.; Horn, M.; Daims, H.;

356

Bartol-Mavel, D.; Wincker, P.; Barbe, V.; Fonknechten, N.; Vallenet, D.; Segurens, B.; Schenowitz-

357

Truong, C.; Medigue, C.; Collingro, A.; Snel, B.; Dutilh, B. E.; Op den Camp, H. J. M.; van der Drift,

358

C.; Cirpus, I.; van de Pas-Schoonen, K. T.; Harhangi, H. R.; van Niftrik, L.; Schmid, M.; Keltjens, J.;

359

van de Vossenberg, J.; Kartal, B.; Meier, H.; Frishman, D.; Huynen, M. A.; Mewes, H. W.; Weissenbach,

360

J.; Jetten, M. S. M.; Wagner, M.; Le Paslier, D., Deciphering the evolution and metabolism of an

361

anammox bacterium from a community genome. Nature 2006, 440, (7085), 790-794.

362

(3) Ma, B.; Wang, S.; Cao, S.; Miao, Y.; Jia, F.; Du, R.; Peng, Y., Biological nitrogen removal from 18

ACS Paragon Plus Environment

Page 18 of 30

Page 19 of 30

Environmental Science & Technology

363

sewage via anammox: Recent advances. Bioresource Technol. 2016, 200, 981-90.

364

(4) Sheng, G. P.; Yu, H. Q.; Li, X. Y., Extracellular polymeric substances (EPS) of microbial aggregates

365

in biological wastewater treatment systems: a review. Biotechnol. Adv. 2010, 28, (6), 882-94.

366

(5) Raszka, A.; Chorvatova, M.; Wanner, J., The role and significance of extracellular polymers in

367

activated sludge. Part I: Literature review. Acta hydrochimica et hydrobiologica 2006, 34, (5), 411-424.

368

(6) Adav, S. S.; Lee, D. J.; Tay, J. H., Extracellular polymeric substances and structural stability of

369

aerobic granule. Water Res 2008, 42, (6-7), 1644-50.

370

(7) Yin, C.; Meng, F.; Chen, G. H., Spectroscopic characterization of extracellular polymeric

371

substances from a mixed culture dominated by ammonia-oxidizing bacteria. Water Res 2015, 68, 740-9.

372

(8) Badireddy, A. R.; Chellam, S.; Gassman, P. L.; Engelhard, M. H.; Lea, A. S.; Rosso, K. M., Role of

373

extracellular polymeric substances in bioflocculation of activated sludge microorganisms under glucose-

374

controlled conditions. Water Res 2010, 44, (15), 4505-16.

375

(9) Strous, M., .; Fuerst, J. A.; Kramer, E. H.; Logemann, S., .; Muyzer, G., .; Pas-Schoonen, K. T., Van

376

De; Webb, R., .; Kuenen, J. G.; Jetten, M. S., Missing lithotroph identified as new planctomycete. Nature

377

1999, 400, (6743), 446-449.

378

(10) Hou, X.; Liu, S.; Zhang, Z., Role of extracellular polymeric substance in determining the high

379

aggregation ability of anammox sludge. Water Res. 2015, 75, 51-62.

380

(11) Meng, F.; Su, G.; Hu, Y.; Lu, H.; Huang, L. N.; Chen, G. H., Improving nitrogen removal in an

381

ANAMMOX reactor using a permeable reactive biobarrier. Water Res 2014, 58, 82-91.

382

(12) Ni, S. Q.; Lee, P. H.; Fessehaie, A.; Gao, B. Y.; Sung, S., Enrichment and biofilm formation of

383

Anammox bacteria in a non-woven membrane reactor. Bioresource Technol. 2010, 101, (6), 1792-9.

384

(13) Tang, C. J.; Zheng, P.; Wang, C. H.; Mahmood, Q.; Zhang, J. Q.; Chen, X. G.; Zhang, L.; Chen, J. 19

ACS Paragon Plus Environment

Environmental Science & Technology

385

W., Performance of high-loaded ANAMMOX UASB reactors containing granular sludge. Water Res

386

2011, 45, (1), 135-44.

387

(14) Ma, Y.; Hira, D.; Li, Z.; Chen, C.; Furukawa, K., Nitrogen removal performance of a hybrid

388

anammox reactor. Bioresource Technol. 2011, 102, (12), 6650-6.

389

(15) Sheng, G.P.; Yu, H.Q.; Li, X.Y., Extracellular polymeric substances (EPS) of microbial aggregates

390

in biological wastewater treatment systems: A review. Biotechnol. Adv. 2010, 28, (6), 882-894.

391

(16) Yu, G. H.; He, P. J.; Shao, L. M.; He, P. P, Stratification Structure of Sludge Flocs with Implications

392

to Dewaterability. Environ. Sci. Technol. 2008, 42, (21), 7944-7949.

393

(17) Li, X. Y.; Yang, S. F., Influence of loosely bound extracellular polymeric substances (EPS) on the

394

flocculation, sedimentation and dewaterability of activated sludge. Water Res 2007, 41, (5), 1022-30.

395

(18) Lin, H.; Zhang, M.; Wang, F.; Meng, F.; Liao, B.-Q.; Hong, H.; Chen, J.; Gao, W., A critical review

396

of extracellular polymeric substances (EPSs) in membrane bioreactors: Characteristics, roles in

397

membrane fouling and control strategies. J. Membrane Sci. 2014, 460, 110-125.

398

(19) Yu, G. H.; He, P. J.; Shao, L. M., Characteristics of extracellular polymeric substances (EPS)

399

fractions from excess sludges and their effects on bioflocculability. Bioresource Technol. 2009, 100, (13),

400

3193-8.

401

(20) Zhang, P.; Fang, F.; Chen, Y. P.; Shen, Y.; Zhang, W.; Yang, J. X.; Li, C.; Guo, J. S.; Liu, S. Y.;

402

Huang, Y.; Li, S.; Gao, X.; Yan, P., Composition of EPS fractions from suspended sludge and biofilm

403

and their roles in microbial cell aggregation. Chemosphere 2014, 117C, 59-65.

404

(21) Jia, F. X.; Yang, Q.; Han, J. H.; Liu, X. H.; Li, X. Y; Peng, Y. Z, Modeling optimization and

405

evaluation of tightly bound extracellular polymeric substances extraction by sonication. Appl. Microbiol.

406

Biot. 2016, 100, (19), 1-10. 20

ACS Paragon Plus Environment

Page 20 of 30

Page 21 of 30

Environmental Science & Technology

407

(22) Li, W. H.; Sheng, G. P.; Liu, X. W.; Yu, H. Q., Characterizing the extracellular and intracellular

408

fluorescent products of activated sludge in a sequencing batch reactor. Water Res 2008, 42, (12), 3173-

409

81.

410

(23) Gabor, R. S.; Burns, M. A.; Lee, R. H.; Elg, J. B.; Kemper, C. J.; Barnard, H. R.; Mcknight, D. M.,

411

Influence of leaching solution and catchment location on the fluorescence of water-soluble organic matter.

412

Environ. Sci. Technol. 2015, 49, (7), 4425-32.

413

(24) Stedmon, C.; Bro, R., Characterizing dissolved organic matter fluorescence with parallel factor

414

analysis: a tutorial. Limnol. Oceanogr.-Meth. 2008, 6, (11), 572–579.

415

(25) Yu, G. H.; He, P. J.; Shao, L. M., Novel insights into sludge dewaterability by fluorescence

416

excitation-emission matrix combined with parallel factor analysis. Water Res 2010, 44, (3), 797-806.

417

(26) K., B., A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric

418

estimation of deoxyribonucleic acid. Biochem. J. 1956, 62, (2), 315-323.

419

(27) Leenheer, J. A.; Croué, J. P., Characterizing aquatic dissolved organic matter. Environ. Sci. Technol.

420

2003, 37, (1), 18A-26A.

421

(28) APHA. Standard Methods for The Examination of Water and Wastewater, 20th ed.; American Public

422

Health Association: Washington, DC, 1999.

423

(29) Pellicer-Nacher, C.; Domingo-Felez, C.; Mutlu, A. G.; Smets, B. F., Critical assessment of

424

extracellular polymeric substances extraction methods from mixed culture biomass. Water Res 2013, 47,

425

(15), 5564-74.

426

(30) Wei, L. L.; Wang, K.; Zhao, Q. L.; Jiang, J. Q.; Kong, X. J.; Lee, D. J., Fractional, biodegradable

427

and spectral characteristics of extracted and fractionated sludge extracellular polymeric substances.

428

Water Res 2012, 46, (14), 4387-96. 21

ACS Paragon Plus Environment

Environmental Science & Technology

429

(31) Sheng, G. P.; Yu, H. Q., Characterization of extracellular polymeric substances of aerobic and

430

anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy.

431

Water Res 2006, 40, (6), 1233-9.

432

(32) McKnight, D. M.; Andersen, D. T., Spectrofluorometric Characterization of Dissolved Organic

433

Matter for Indication of Precursor Organic Material and Aromaticity. Limnol. Oceanogr. 2001, 46, (1),

434

38–48.

435

(33) Y. Z.; Zhang, E.; Yin, Y.; Dijk, M. A. V.; Feng, L.; Shi, Z.; Liu, M.; Qina, B., Characteristics and

436

sources of chromophoric dissolved organic matter in lakes of the Yungui Plateau, China, differing in

437

trophic state and altitude. Limnol. Oceanogr. 2010, 55, (6), 2645-2659.

438

(34) Yuan, D. Q.; Wang, Y. L.; Feng, J., Contribution of stratified extracellular polymeric substances to

439

the gel-like and fractal structures of activated sludge. Water Res 2014, 56, 56-65.

440

(35) Vlaeminck, S. E.; Terada, A.; Smets, B. F.; De Clippeleir, H.; Schaubroeck, T.; Bolca, S.;

441

Demeestere, L.; Mast, J.; Boon, N.; Carballa, M.; Verstraete, W., Aggregate size and architecture

442

determine microbial activity balance for one-stage partial nitritation and anammox. Appl. Environ.

443

Microb. 2010, 76, (3), 900-9.

444

(36) Kartal, B.; Maalcke, W. J.; de Almeida, N. M.; Cirpus, I.; Gloerich, J.; Geerts, W.; Op den Camp,

445

H. J.; Harhangi, H. R.; Janssen-Megens, E. M.; Francoijs, K. J.; Stunnenberg, H. G.; Keltjens, J. T.; Jetten,

446

M. S.; Strous, M., Molecular mechanism of anaerobic ammonium oxidation. Nature 2011, 479, (7371),

447

127-30.

448

(37) Snook, C. F.; Tipton, P. A.; Beamer, L. J., Crystal Structure of GDP-Mannose Dehydrogenase:  A

449

Key Enzyme of Alginate Biosynthesis in P. aeruginosa. Biochemistry 2003, 42, (16), 4658-4668.

450

(38) Lin, Y. M,; de Kreuk, M.; van Loosdrecht, M. C.; Adin, A., Characterization of alginate-like 22

ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30

Environmental Science & Technology

451

exopolysaccharides isolated from aerobic granular sludge in pilot-plant. Water Res 2010, 44, (11), 3355-

452

64.

453

(39) Flemming, H. C.; Wingender, J., The biofilm matrix. Nat. Rev. Microbiol. 2010, 8, (9), 623-33.

454

(40) Davis, T. A.; Volesky, B.; Mucci, A., A review of the biochemistry of heavy metal biosorption by

455

brown algae. Water Res. 2003, 37, (18), 4311-4330.

456

(41) Chen, W. P.; Chen, J. Y.; Chang, S. C.; Su, C. L., Bacterial Alginate Produced by a Mutant of

457

Azotobacter vinelandii. Appl. Environ. Microb. 1985, 49, (3), 543-6.

458

(42) Zhang, L.; Feng, X.; Zhu, N.; Chen, J., Role of extracellular protein in the formation and stability

459

of aerobic granules. Enzyme Microb. Tech. 2007, 41, (5), 551-557.

460

(43) Liu, Y. Q.; Liu, Y.; Tay, J. H., The effects of extracellular polymeric substances on the formation

461

and stability of biogranules. Appl. Microbiol. Biot. 2004, 65, (2), 143-148.

462

(44) Fenderson, Bruce. Molecular Biology of the Cell,5rd, ed.; Garland Science: New York, 2008.

463

(45) Tang, X.; Liu, S. T.; Zhang, Z. T.; Zhuang, G. Q., Identification of the release and effects of AHLs

464

in anammox culture for bacteria communication. Chem. Eng. J. 2015, 273, 184-191.

465

(46) Wan, C.; Lee, D. J.; Yang, X.; Wang, Y.; Wang, X.; Liu, X., Calcium precipitate induced aerobic

466

granulation. Bioresource Technol. 2015, 176, 32-7.

467

(47) Duan, X.; Zhou, J.; Qiao, S.; Wei, H., Application of low intensity ultrasound to enhance the activity

468

of anammox microbial consortium for nitrogen removal. Bioresource Technol. 2011, 102, (5), 4290-3.

469

(48) Liu, S. T.; Yang, F. L.; Meng, F. G.; Chen, H. H.; Gong, Z., Enhanced anammox consortium activity

470

for nitrogen removal: impacts of static magnetic field. J. Biotechnol 2008, 138, (3-4), 96-102.

471

(49) Verrier, D.; Mortier, B.; Albagnac, G., Initial adhesion of methanogenic bacteria to polymers.

472

Biotechnol. Lett. 1987, 9, (10), 735-740. 23

ACS Paragon Plus Environment

Environmental Science & Technology

473

(50) van Loosdrecht, M. C.; Lyklema, J.; Norde, W.; Schraa, G.; Zehnder, A. J., The role of bacterial cell

474

wall hydrophobicity in adhesion. Appl. Environ. Microb. 1987, 53, (8), 1893.

475 476 477

TABLE AND FIGURE CAPTIONS

478

Table 1 Band assignments for FTIR spectral features (cm-1) of slime and TB-EPS. The

479

band assignments are based on previous reports 7, 8, 10, 38.

480

Fig. 1 Characterization of EPS fractions (a) colorimetric components. EEM contours

481

of three components (b) Component 1, (c) Component 2 and (d) Component 3

482

decomposed using the PARAFAC approach. (e) fluorescence components in the

483

different anammox sludges. The line in the middle of the box marks the median and the

484

star point marks the mean. The boundary of the box indicates the 25th percentile and

485

the 75th percentile. Whiskers that protrude out of the box indicate the 10th and 90th

486

percentiles. The hollow and solid circle point represent the minimums and maximums.

487

The value above solid circle is the mean and the standard deviation (in parentheses).

488

Fig. 2 PCA ordination diagram based on compositional characteristics of three spatial

489

scales of EPS. (a) slime; (b) LB-EPS; (c) TB-EPS. Different groupings is enclosed in

490

polygon based on sludge morphology (a-2, b-2, c-2) and water quality (a-3, b-3, c-3).

491

Fig. 3 Pearson correlation analysis between major genera and EPS parameters.

492

Heatmap analysis of (a) slime, (b) LB-EPS and (c) TB-EPS. The strength of correlation

493

is defined by a color code with red indicating positive correlations, white a neutral

494

context, and blue a negative correlation. (Ratio=α-helix/(β-sheet + random coil), white

495

stars, p < 0.05; black stars, p < 0.01). 24

ACS Paragon Plus Environment

Page 24 of 30

Page 25 of 30

Environmental Science & Technology

496

Fig. 4 Pearson correlation analysis of anammox abundance with UA in slime (a-1) and β-sheet in

497

TB-EPS (b-1). FTIR spectra of slime (a-2) and TB-EPS (b-2). (c) The primary mechanisms of

498

anammox aggregation behavior (the pictures of protein secondary structures was cited from

499

Molecular Biology of the Cell,5th 44 )

25

ACS Paragon Plus Environment

Environmental Science & Technology

Table 1 Band assignments for FTIR spectral features (cm-1) of slime and TB-EPS. The band assignments are based on previous reports 7, 8, 10, 38. Wavenumber (cm-1) slime

TB-EPS

3323 2927 1655 1540 1402 1240

3393 2937 1668 1623 1384 -

1158

-

1075 952 859

1112 1046, 995 -

Band assignments O-H stretching (hydrogen-bonded) ν C-H stretching (-CH2 and -CH3 groups) νas COO- stretches possibly associated with uronic acid ν C=C stretch associated with proteins νs C=O stretch (amide I) associated with proteins ν C=C stretches possibly associated with pyranose ring νs COO- stretches possibly associated with uronic acid δ C-H stretches in -CH3 associated with amines and lipids νs C-N stretch possibly associated with O-acetyl ester δ C-OH, δ C-O and ν C-O possibly associated with polysaccharide Ring vibrations ν P=O, ν C-O-C, ν C-O-P as in polysaccharides δ C-H stretching (-CH groups) νas O-P-O stretches associated with nucleic acids Ring “breathing” associated with ν C-C and ν C-OH

500

26

ACS Paragon Plus Environment

Page 26 of 30

Environmental Science & Technology

0.12 (0.15)

0.8

350

0.08 (0.31)

2.35 (4.21)

14.20 (5.91)

0.48 (0.77)

2.25 (2.03)

0.4

8.60 (4.40)

8.55 2.82 (1.51) (2.00) 1.21 (1.23)

0.03 (0.02)

0.2

0.3

DNA

300

HA

UA

DOC

UV 254

0.02

250

LB

0.005 200

(b) (c)

0.0 300

350

400 450 Em (nm)

500

550

200

0.045

Component 2

0.03 300

0.025

8

0.005 200

16.51

2.0

(29.08)

100

1.5

19.86

11.10 10.39

(21.78)

(16.04) (15.88)

(c) (d)

300

350

0.49 (0.16)

1.0

400 450 Em (nm)

500

0.2

0.0

550

200 x 10 18

400 Component 3

250

300

350

400

Wavelength (

-3

0.3 Component 3

16 14

350

0.2

12 Ex (nm)

(0.13) 1.59 1.59 (0.12) (0.11)

FI & HIX

6 1.72

0.3

0.1

0.01

(1.41)

27.40

0.4

0.015

23.89

(43.20)

400

Component 2

0.5

0.035

250

(42.14)

350

Wavelength (

(1.72) 1.88

(62.96)

300

0.04

SUVA

1.77

250

400

350

TB

53.09

250

0.2

0.1

0.01

0.02

Slime

163.48

150

300

0.015

0.0

PS

0.03 0.025

Ex (nm)

2.17

20 (5.33)

0.69 (1.96) 2.99 (2.33)

Component 1

0.4

0.035

10

300

8

Loadings

40

200

Component 1

Loadings

1.0

400

Loadings

0.62 (0.12)

0.6

(55.25)

0.1 6

50

250

5.98

2

0

0.0

C1

C2

C3

4

0.5

(1.60)

501

1.4

(a) (b)

Ex (nm)

0.64 (0.16)

22.92 (19.10)

8.02 (12.88)

30.40 (9.98)

1.6

1.2

80 60

0.39 0.55 (0.31) (0.41)

-1

Concentration (mg/g VSS)

45.09 (21.63)

56.32 (15.42)

PN

Fluorescence intensity scores

103.62 (14.52)

TB

100

0

(e)

LB

-1

Slime

120

-1

(a)

m mg ) UV254(m ) & SUVA(L·

Page 27 of 30

FI

HIX

200

0.0 300

350

400 450 Em (nm)

500

550

502

Fig. 1 Characterization of EPS fractions (a) colorimetric components. EEM contours of three

503

components (b) Component 1, (c) Component 2 and (d) Component 3 decomposed using the

504

PARAFAC approach. (e) fluorescence components in the different anammox sludges. The line

505

in the middle of the box marks the median and the star point marks the mean. The boundary

506

of the box indicates the 25th percentile and the 75th percentile. Whiskers that protrude out of

507

the box indicate the 10th and 90th percentiles. The hollow and solid circle point represent the

508

minimums and maximums. The value above solid circle is the mean and the standard

509

deviation (in parentheses).

27

ACS Paragon Plus Environment

200

250

300

350

400

Wavelength (

Environmental Science & Technology

(a-1)

Page 28 of 30

(c-1)

(b-1)

UV254

μanammox μNOB

UV254

μanammox

UV254

Slime

LB-EPS

(a-2)

(b-2)

Slime

LB-EPS

(a-3)

(b-3)

Slime

μAOB μNOB

μNOB μAOB

μAOB

μanammox

TB-EPS

(c-2)

TB-EPS

(c-3)

TB-EPS

LB-EPS

510 511

Fig. 2 PCA ordination diagram based on compositional characteristics of three spatial scales

512

of EPS. (a) slime; (b) LB-EPS; (c) TB-EPS. Different groupings is enclosed in polygon based

513

on sludge morphology (a-2, b-2, c-2) and water quality (a-3, b-3, c-3).

28

ACS Paragon Plus Environment

Page 29 of 30

Environmental Science & Technology

(a) 0.8 Nitrospira AOB Anammox Nitrobacter DNB (nirK) Total bacterial DNB (nirS)

0.6 0.4 0.2 0 -0.2

C2 C1 β-sheet FI α-Helix HIX PS DOC UA Random coil C3 Ratio β-turn HA DNA UV254 PN SUVA

-0.4

(b) 0.4 Nitrospira AOB Total bacterial DNB(nirS) DNB(nirK) Nitrobacter Anammox

0.2 0 -0.2 -0.4

Ratio α-Helix β-turn FI C2 C3 C1 UV254 DNA SUVA HA PS β-sheet DOC HIX PN Random coil UA

-0.6

(c) 0.8 Anammox Total bacterial DNB (nirS) Nitrospira AOB Nitrobacter DNB (nirK)

PN UA PS DNA DOC HA SUVA UV254 C2 HIX β-sheet Ratio α-Helix FI C3 Random coil C1 β-turn

514

0.6 0.4 0.2 0 -0.2 -0.4

515

Fig.3 Pearson correlation analysis between major genera and EPS parameters. Heatmap

516

analysis of (a) slime, (b) LB-EPS and (c) TB-EPS. The strength of correlation is defined by a

517

color code with red indicating positive correlations, white a neutral context, and blue a

518

negative correlation. (Ratio=α-helix/(β-sheet + random coil), white stars, p < 0.05; black stars,

519

p < 0.01).

29

ACS Paragon Plus Environment

Environmental Science & Technology

(a-1) 7 6

(a-2) 100

Anammox ∝ UA (slime) -10

Page 30 of 30

Slime (sample G4/S for exemple)

90

2

y=1.7233×10 x+2.1312 (R =0.7259) Transmittance (q.u.)

1240

UA (mg/L)

5 4 3

80

70

60

Linear fit 95% Confidence bands

1 0.00E+000

5.00E+009

1.00E+010

1.50E+010

2.00E+010

40

2.50E+010

3500

1158 C-O 1075 C-O-C

1655 COO

3323 O-H

4000

3000

952 O-P-O

1540 C=C

2927 C-H

50

2

859 C-C C-OH

1402 COO

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Anammox abundance (copies/mg-dry sludge)

β-sheet (%)

35

(b-2) 100

Anammox ∝ β-sheet (TB-EPS) -10

2

TB-EPS (sample G4/S for exemple)

90

y=7.3389×10 x+17.7060 (R =0.6436) Transmittance (q.u.)

(b-1) 40

30

25

20

80 2937 C-H

70

60

2555 S-H

Linear fit 95% Confidence bands

100

995 1046 C-H C-H

3393 O-H

90

1668 C=C 1623 C=O

50

15

1112 C-O-C

80

1384 CH3

40

70

0.00E+000

5.00E+009

1.00E+010

1.50E+010

2.00E+010

2.50E+010

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Anammox abundance (copies/mg-dry sludge)

60

50

(c)

β-Sheet

α-Helix 40

4

hydrophobic grouping

(–G–)n

(–MG–)n

(–M–)n

(–G–)n

520 521

Fig. 4 Pearson correlation analysis of anammox abundance with UA in slime (a-1) and β-sheet

522

in TB-EPS (b-1). FTIR spectra of slime (a-2) and TB-EPS (b-2). (c) The primary mechanisms

523

of anammox aggregation behavior (the pictures of protein secondary structures was cited from

524

Molecular Biology of the Cell,5th 44 ).

30

ACS Paragon Plus Environment