Characteristic Regions of the Fluorescence ExcitationâEmission

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Environmental Measurements Methods

Characteristic Regions of Fluorescence ExcitationEmission Matrix (EEM) to Identify Hydrophobic/Hydrophilic Contents of Organic Matter in Membrane Bioreactors Kang Xiao, Yuexiao Shen, Shuai Liang, Jihua Tan, Xiao-Mao Wang, Peng Liang, and Xia Huang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02684 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 9, 2018

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Environmental Science & Technology

1

Characteristic Regions of Fluorescence Excitation-Emission Matrix

2

(EEM) to Identify Hydrophobic/Hydrophilic Contents of Organic

3

Matter in Membrane Bioreactors

4 5

Kang Xiaoa,b, Yuexiao Shenc,*, Shuai Liangd, Jihua Tana, Xiaomao Wangb,

6

Peng Liangb, Xia Huangb,*

7 a

8

College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China

9 10

b

State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing 100084, China

11 c

12 13

d

Department of Chemistry, University of California, Berkeley, CA 94720, USA

College of Environmental Science and Engineering, Beijing Forestry University, Beijing 100083, China

14 15 16 17 18

___________________________________________________________________________

19

*Corresponding authors

20

E-mail addresses: [email protected] (Y. Shen), [email protected] (X. Huang).

21

1

ACS Paragon Plus Environment


22 23

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ABSTRACT: This study systematically investigated the correlations between fluorescence distributions

24

characterized

by

excitation-emission

matrix

(EEM)

25

composition of dissolved organic matter (DOM) in membrane bioreactors (MBRs). Based on

26

samples from 10 full-scale MBRs, we performed point-to-point comparisons among different

27

components using an EEM fluorescence quotient (FQ) method, and obtained a

28

hydrophobic/hydrophilic fluorophore distribution map via Wilcoxon signed rank test.

29

Hydrophobic acids/bases (HOA/HOB) concentrated in the low-wavelength region (excitation

30

wavelength Ex < 235 nm), while hydrophilic substances (HIS) were enriched in the region of

31

Ex > 235 nm (especially with emission wavelength Em = 300-360 nm). Quantitatively, EEM

32

regional contribution to whole-wavelength fluorescence was found to significantly correlate

33

with the hydrophobic/hydrophilic proportions of DOM, with Pearson’s coefficients of 0.94

34

and 0.78 (p < 0.01) for HOA and HIS, respectively. We established a linear regression model

35

showing HOA proportion as a function of EEM regional contribution at (Ex, Em) = (200-285,

36

340-465) nm with R2 = 0.876 which was validated via leave-one-out cross-validation and

37

Monte Carlo simulation. This study shows a statistically hydrophobicity-dependent

38

fluorescence property across different MBRs, and it might be applied to provide a quick

39

estimation of hydrophobic/hydrophilic composition of DOM in wastewater treatment systems

40

based on EEM monitoring.

41

2


and

hydrophobic/hydrophilic

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42

TOC/Abstract Art

43 44

3



45

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1. Introduction

46

Hydrophobicity is a basic property of dissolved organic matter (DOM) in wastewater

47

treatment systems 1. Hydrophobic interaction intervenes in DOM’s basic interfacial behaviors

48

such as adsorption and phase partitioning 2, and could exert profound impacts on migration

49

and transformation behaviors in wastewater treatment systems 1. Taking membrane bioreactor

50

(MBR) that has developed rapidly in recent years 3 as an example, hydrophobicity may affect

51

multiple aspects including: (a) DOM/membrane interaction on membrane fouling 4; (b)

52

DOM/biomass interaction that may not only reflect microbial status 5but also influence

53

sludge flocculation and sedimentation 6; (c) DOM/gas interaction on oxygen transfer during

54

aeration 7; (d) DOM/solute interaction on elimination of trace organics 8; and (e) removal of

55

DOM in physical/chemical processes such as adsorption, coagulation and oxidation

56

Therefore, monitoring of DOM’s hydrophobic/hydrophilic composition would provide

57

extensive implications for comprehensive understanding and targeted control of wastewater

58

treatment processes.

59

9,10

.

Adsorptive resin column chromatography is a textbook method for characterizing DOM’s 11

60

hydrophobic/hydrophilic composition

61

(e.g.

62

(HOA/HOB/HON) and hydrophilic substances (HIS), and HIS can be further classified into

63

hydrophilic acids/bases/neutrals (HIA/HIB/HIN) using ion exchange resins

64

enabling a classical definition of the hydrophobic/hydrophilic components, the whole

65

procedure is cumbersome. The fractionation process takes 12-36 h, and pretreatment of the

66

resins (including Soxhlet extraction of impurities) requires extra 3-7 d 12,13.

DAX

or

XAD)

fractionates

. Under certain pH conditions, the adsorptive resin DOM

into

hydrophobic

4


acids/bases/neutrals

11

. While

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67

Excitation-emission matrix (EEM) fluorescence spectroscopy may be a potential candidate

68

for fast detection of hydrophobic/hydrophilic composition. EEM is a convenient and sensitive

69

method providing vast amount of fingerprinting information for DOM characterization

70

Distribution of fluorescence over different EEM wavelength regions has been extensively

71

found to be indicative of DOM’s chemical composition

72

biodegradability

73

formation 18, and fate in wastewater treatment processes19. In order to fulfill the potential of

74

EEM for indicating DOM’s hydrophobicity, it is critical to establish any qualitative or

75

quantitative relationship between fluorescent signals and hydrophobic/hydrophilic contents.

76

This has been pioneered by a few researchers in their case studies. Qualitatively, it is

77

commonly recognized that the distribution of fluorescence peaks on the EEM map differs

78

between hydrophobic/hydrophilic fractions of DOM

79

that DOM fluorophores from different EEM wavelength regions had different degrees of

80

hydrophobicity, when detecting EEM spectra of DOM fractions eluted from a C18

81

reversed-phase adsorptive column 22. Xiao et al. found that HIS had lower quantum yield and

82

smaller Stokes shift than did hydrophobic components (HOA and HOB), with its

83

fluorescence more prone to peak in the EEM region where the emission wavelength was

84

close to the excitation wavelength

85

content was significantly related to Peak C position or Peak T intensity of EEM in a case

86

study of surface water

87

not been sufficiently reported in the field of wastewater treatment.

17

, trace of biological activity

16

14,15

, humification degree

.

17

,

17

, potential of disinfection by-product

20,21

. More specifically, Li et al. found

23

. Quantitatively, Baker et al. found that the hydrophilic

24

. However, to our knowledge, such quantitative relationships have

5



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88

This study aims to explore the dependence of EEM regional fluorescence distribution on

89

hydrophobic/hydrophilic composition, by investigating DOM samples from 10 full-scale

90

MBR wastewater treatment plants (WWTPs). At the qualitative level, characteristic regions

91

for HIS/HOA/HOB on the EEM map were identified using a fluorescence quotient (FQ)

92

approach combined with nonparametric statistical validation. At the quantitative level, the

93

characteristic regions that can predict the hydrophobic/hydrophilic contents of DOM were

94

explored via correlation analysis, and Monte Carlo simulations were conducted to evaluate

95

the robustness of the regression models. This study may provide new clues for rapid

96

indication of DOM hydrophobic/hydrophilic composition using fluorescence spectroscopy.

97 98

2. Materials and Methods

99

2.1. DOM Samples

100

DOM samples were obtained from 10 full-scale MBR-based municipal WWTPs, each with

101

a designed capacity of over 20,000 m3/d. These plants are geographically distributed over

102

China (Central, North, East and Southwest China), involved in different basins (Yangtze

103

River, Hai River, Tai Lake and Dian Lake) and a mixture of metropolitan areas and countries.

104

Thus the resulting DOM samples were diverse and considered to be representative for typical

105

municipal

106

anaerobic/anoxic/aerobic-membrane bioreactor (AAO-MBR) and its variants, as shown in

107

Table S1 in Supporting Information. Some properties of the influent, effluent and mixed

108

liquor are listed in Table S2. At the time of sampling, all the WWTPs (except the #3 WWTP

109

undergoing sludge bulking) had been stably operated for at least one year.

WWTP

systems.

The

process

configurations

6


of

the

WWTPs

are

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110

The sampling was undertaken from late October to next January. At each WWTP, sludge

111

suspensions were collected from three parallel corridors of membrane tanks as three replicas.

112

The sludge suspension was filtered on-site using a qualitative filter paper and a GF/F

113

glass-fiber filter (Whatman, UK) sequentially to obtain DOM as the filtrate. The DOM

114

samples were stored in a 4 °C coolbox and immediately conveyed to laboratory (in 24 h) for

115

subsequent analyses.

116

2.2. Fractionation of Hydrophobic/Hydrophilic Components

117

Through adsorptive column chromatography, DOM was fractionated into hydrophobic

118

acids/bases/neutrals (HOA/HOB/HON) and hydrophilic substances (HIS). Supelite DAX-8

119

resin (Supelco, USA) used for the fractionation has a particle size of 40-60 mesh, an average

120

pore size of 22.5 µm, a specific surface area of 160 m2/g, and a pore volume of 0.79 cm3/g.

121

The plexiglass column has a dimension of Φ 1.0 cm × 20 cm, with a dead volume of 10 mL.

122

The critical retention factor for the column chromatography was 25. The fractionation

123

procedure is illustrated in Figure S1 in Supporting Information. HOA is adsorbed by the resin

124

at lower pH (pH = 2) and released at higher pH (0.1 M NaOH); HOB is adsorbed at neutral

125

pH and released at lower pH (0.1 M HCl); HIS is immune to adsorption while HON is

126

adsorbed at all pHs. These fractions, as obtained, were diluted back to have the same volume

127

as that of the original water sample, and re-adjusted to neutral pH. Considering that HON

128

containing a considerable amount of methanol that could interfere with fluorescence 25, HON

129

was excluded from subsequent measurements. Total organic carbon (TOC) concentrations of

130

the fractions were determined using a TOC analyzer (TOC-VCPH, Shimadzu, Japan).

131

2.3. EEM Measurement 7



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132

EEM fluorescence spectra of the total DOM and its hydrophobic/hydrophilic components

133

were measured using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). Prior to the

134

measurements, all the samples were adjusted to pH 7.0 ± 0.1 using 0.01 M HCl or NaOH.

135

The ionic strength was not adjusted considering its minimal impact on fluorescence

136

fluorescence was scanned at a speed of 2400 nm/min in the wavelength range of (Ex, Em) =

137

(200-450, 250-550) nm, with a slit width of 5 nm and a photomultiplier voltage of 700 V. The

138

obtained EEM data were then treated by subtracting water background, eliminating first- and

139

second-order Rayleigh and Raman scattering signals using an interpolation method

140

correcting the inner-filter effect using the same sample’s UV-Vis absorbance in the

141

wavelength range of 200-550 nm (the maximum absorbance was smaller than 1.5 which may

142

ensure the effectiveness of the correction)

143

intensity using water’s first-order Raman peak area as a reference, such that the resultant

144

intensity was expressed in Raman Unit (R.U.) 30.

145

2.4. Fluorescence Quotient (FQ) Method

146

28,29

26

. The

27

,

, and standardizing the unit of fluorescence

The differences in EEM spectra between different hydrophobic/hydrophilic components 31

147

were identified using a fluorescence quotient (FQ) method

148

steps. First, fluorescence intensity (FI) of EEM spectra was normalized and converted to

149

relative fluorescence intensity (FI'):

150

FI′ = 0.01 + 0.99 ×

that was implemented in two

FI − FI min FI max − FI min

(1)

151

where FImax and FImin denote the maxium and minimum fluorescence intensities respectively

152

over the whole wavelength range. Second, FQ is calculated as the element-wise matrix

153

division of FI' values between two EEM spectra (A and B) with logarithmization: 8


Page 9 of 27


154

FQ A:B = log10 (FI′A / FI′B )

155

At a certain wavelength position (Ex, Em), FQA:B > 0 indicates that A has a higher relative

156

intensity than B at this point, and vice versa. FQHOA:HIS, FQHOB:HIS and FQHOA:HOB were thus

157

obtained to identify the relative abundance of different hydrophobic/hydrophilic components

158

for fluorescence distribution on the EEM map. Statistics of FQs were performed over the

159

samples from the 10 WWTPs. One-tailed non-parametric Wilcoxon signed rank test was

160

employed to judge whether the FQ at each wavelength position was significantly positive or

161

negative. Significance level p < 0.01 means highly significant, 0.01 ≤ p < 0.05 means

162

significant, 0.05 ≤ p < 0.1 means moderately significant, and p ≥ 0.1 means insignificant. The

163

qualitative identification of +/− signs (with statistical confidence) is regarded as a primary

164

step of a series of qualitative/quantitative analyses 32.

165

2.5. Scanning EEM Regions for Fluorescence Correlation

(2)

166

Given a certain position (Exi, Emi), regional fluorescence proportion (fi) is defined as the

167

ratio of the sum of fluorescence intensity in the neighborhood region (Exi ± ∆λ, Emi ± ∆λ) to

168

that in the entire wavelength range of (Ex, Em) = (200-450, 250-550) nm:

fi =

169

∑ ∑

FI

Ex i ±∆λ Emi ±∆λ

∑ ∑

FI

(3)

Any Ex Any Em

170

where ∆λ is the span of the neighborhood. In principle, fi reflects the relative number

171

distribution of photons with varied energy grades (and hence distribution of fluorophores).

172

Indeed, the concept of regional fluorescence proportion or relative fluorescence has been well

173

employed in the fluorescence regional integration and parallel factor analysis of EEM spectra

174

16,17,28

175

scanned (as illustrated in Figure S2 in Supporting Information), and was correlated with

. Using a self-made MATLAB R2015b program, fi at all positions with varied ∆λ’s was

9



176

hydrophobic/hydrophilic contents over the DOM samples from the 10 WWTPs. Pearson’s

177

correlation coefficient ri for each (Exi ± ∆λ, Emi ± ∆λ) region was logged during the scan. For

178

the regions representing highly significant correlation, linear regression was conducted to

179

further seek quasi-quantitative relationships that were assessed using R2, F-test significance at

180

95% confidence intervals (of mean and individual) and leave-one-out cross-validation Q2.

181

The robustness of the regression model was examined using 10,000 times of Monte Carlo

182

simulation based on t-distribution of the mean-square error of the model.

183 184

3. Results and Discussion

185

3.1. Overview of Hydrophobic/Hydrophilic DOM Components from the 10 MBRs

186

The concentrations of the total DOM and its hydrophobic/hydrophilic proportions in the 10

187

WWTPs are given in Table 1. The total TOC concentrations were distributed from 6.6 to 22.2

188

mg/L. The TOC proportions of HIS, HOA, and HOB varied in the ranges of 41%-59%,

189

24%-33%, and 5%-14%, respectively. The total DOM as well as the hydrophobic/hydrophilic

190

composition exhibited a certain degree of diversity among the 10 WWTPs.

191

The EEM spectra of DOM and its hydrophilic/hydrophobic components are shown in

192

Figure 1. Among the 10 WWTPs, the EEM profiles (e.g. contour shape and regional intensity)

193

were not all the same. It also differed among the hydrophobic/hydrophilic components (HIS,

194

HOA and HOB). EEM of the total DOM appeared to be a mixture of EEMs of the

195

components. The overall fluorescence densities (in the whole wavelength range) for HIS,

196

HOA and HOB were 0.035±0.011, 0.083±0.023 and 0.135±0.089 R.U./(mg/L), respectively.

197

Polysaccharide/protein/humic contents in HIS, HOA and HOB are provided in Table S3 in 10


Page 10 of 27

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198

Supporting Information. Slight shifts (~10 nm) of fluorescence peak positions can be

199

observed when comparing the EEMs of HOA and HOB with that of the total solution in the

200

wavelength range of Ex < 250 nm and Em < 400 nm. The acid/base treatment in the current

201

fractionation procedure (Figure S1) may have altered the secondary, tertiary or quaternary

202

structure of the macromolecules to some extent

203

processes such as vibrational relaxation to induce peak shifts 34. Nonetheless, the slight peak

204

shifts were considered to have no substantial impact on the analyses in the subsequent

205

sections.

33

, and hence influence fluorescence

206 207

Table 1 Total TOC concentrations of membrane tank DOM and the hydrophobic/hydrophilic

208

components from the 10 WWTPs.a Total

concentration

WWTP

HIS proportion

HOA proportion

HOB proportion

(mg-TOC/L)

209

#1

9.2 (±0.4)

59% (±5%)

33% (±2%)

5% (±5%)

#2

13.8 (±0.8)

59% (±2%)

32% (±2%)

10% (±1%)

#3

22.2 (±1.1)

58% (±3%)

24% (±1%)

6% (±1%)

#4

11.5 (±1.2)

57% (±6%)

28% (±2%)

7% (±2%)

#5

8.5 (±0.1)

55% (±4%)

31% (±1%)

11% (±1%)

#6

9.6 (±0.4)

52% (±5%)

31% (±2%)

11% (±1%)

#7

6.6 (±0.2)

44% (±2%)

31% (±1%)

14% (±1%)

#8

7.7 (±0.9)

43% (±3%)

32% (±4%)

14% (±2%)

#9

13.3 (±0.4)

43% (±2%)

26% (±2%)

5% (±0%)

#10

12.6 (±0.4)

41% (±3%)

25% (±1%)

9% (±1%)

a

Data given as average (± standard deviation) according to triplicate measurements.

210

11



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211 Plants: #1

#2

#3

#4

#5

#6

#7

#8

#9

#10

Total HIS

450

450

400

400

350

350

300

300

250

250

200 450

200 450

400

400

350

350

300

300

250

250

250 350 450 550 450 400 350 300 250 200

Ex (nm)

Em (nm)

200 200 250 300 350 400 450 500 550 250 300 350 400 450 500 550 250 300 350 400 450 500 550 250 300 350 400 450 500450 550 250 300 350 400 450 500450 550 250 300 350 400 450 500 550 250 300 350 400 450 500 550 250 300 350 400 450 500 550 250 300 350 400 450 500 550 250 300 350 400 450 500 550 400 400

HOA HOB 0 1.2 2.4 0

212 213

5

350

350

300

300

250

250

200 450

200 450

400

400

350

350

300

300

250

250

200

200

10 0 3.6 7.2 0 1.2 2.4 0

5

10 0

5

10 0 1.2 2.4 0 1.2 2.4 0 3.6 7.2 0

5

10

Intensity (R.U.)

Figure 1. EEM spectra of DOM hydrophobic/hydrophilic components from the 10 MBRs.

214 215

3.2. Qualitative Identification of Characteristic Regions of EEM

216

Fluorescence quotient (FQ) was used to systematically identify the differences in EEM

217

spectra among the hydrophobic/hydrophilic components. The FQ contours were depicted on

218

an EEM map (Figure S3 in Supporting Information), from which the distribution of FQ in

219

different wavelength regions can be clearly observed. The positive FQHOA:HIS and FQHOB:HIS

220

strongly peaked in the lower Ex region (Ex < 235 nm), and the positive FQHOA:HOB peaks

221

mainly distributed in the middle Em region (Em = 350-450 nm). The significance of positive

222

or negative FQ at each (Ex, Em) point was verified using Wilcoxon signed rank test, with the

223

results plotted in Figure 2. FQHOA:HIS and FQHOB:HIS were significantly larger than 0 in the

224

region of Ex < 235 nm (at the bottom of EEM), indicating that hydrophobic fluorophores are

225

more prone to occur in this region. FQHOA:HIS and FQHOB:HIS were significantly negative in the

226

region where Em is not far from Ex (i.e. close to the 45° sloping border of EEM, especially in

227

Figure 2a), indicating that hydrophilic fluorophores tend to appear in this region. The close 12


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228

values of Em and Ex correspond to a small Stokes shift and hence a small energy gap

229

between excitation and emission lights 23.

230

Based on the above results, Figure 3a provides a simple sketch of the characteristic regions

231

of hydrophobic/hydrophilic components on the EEM map, and is compared with the classical

232

regions in terms of chemical species (Figure 3b)

233

235 nm or Em < 300 nm) represent strong propensity for HOA or HOB (Figure 3a), and

234

might correspond to aromatic protein-like and fulvic acid-like substances (Figure 3b).

235

Hydrophobic components are well reported to bear polycyclic phenols/acids and/or aromatic

236

amines

237

between HOB and fulvic acids. The region of (Ex > 235 nm and Em = 300-360 nm)

238

represents strong occurrence of HIS (Figure 3a), and may correspond to soluble microbial

239

by-product-like species or Peak B/T-related fluorophores (Figure 3b). In principle, HIS’s

240

fluorescence also arises from π-conjugated fluorophores, but the plenty of hydrophilic groups

241

(e.g. -OH), as substituents or interferring neighbors of the fluorophores

242

characteristic region of HIS different from those of the hydrophobic components. The region

243

of (Ex > 235 nm and Em = 380-450 nm) has traces of both HIS and HOA (Figure 3a), and

244

pertains to humic acid-like substances (Figure 3b). It is likely that HIS and HOA share some

245

molecular segments 12,35, such that their fluorescence overlaps in this region. For reference, it

246

was recently reported that the fluorescence intensities in the regions that correspond to the

247

“HIS > HOA” and “HOA~HOB” regions in Figure 3a were negatively correlated in the case

248

of landfill leachate DOM 36. It was also shown from asynchronous correlation of fluorescence

249

spectra that the peaks of tyrosine-, tryptophan-, fulvic- and humic-like substances can all vary

16,17

. The lower-wavelength regions (Ex
450 nm) reveals mismatch

13


34

, may render the


Page 14 of 27

37

250

with hydrophobicity or polarity of the DOM fractions

. Chemical compositions of HIS,

251

HOA and HOB in this study are provided in Table S3 in Supporting Information. In the

252

context of full-scale MBRs, it was reported that DOM in the region of (Ex, Em) = (200-280,

253

280-330) nm had a high propensity for membrane fouling, and that in the region of (Ex, Em)

254

= (200-320, 380-500) nm had a moderate propensity for fouling

255

regions with Figure 3a suggests that HIS, HOA and HOB all pose potential risk of fouling.

31

. A comparison of these

256 257

258 259

Figure 2. Statistically significant regions for the positive and negative fluorescence quotients

260

(FQs) between different hydrophobic/hydrophilic components according to Wilcoxon signed

261

rank test (sample numer n = 10).

262

14


Page 15 of 27


263 (a)

(b)

450

450 Regions by Chen et al. (2003) Peaks by Coble et al. (2014)

400

400 Uncertain

Peak C (humic-like) or Peak α (older organics)

350

Ex (nm)

Ex (nm)

350 HIS > HOA 300

300

Soluble microbial Humic acid-like by-product-like Peak M (marine humic-like) Peak B (tyrosine-like) or Peak β (freshly produced organics)

HIS 250

HOB > HOA

HIS ~ HOA HOB > HOA

HOA ~ HOB 200 250

300

250

350

450

500

200 250

550

300

350

Em (nm)

264

Peak A (humic-like)

Aromatic Aromatic protein I protein II

HOA > HOB 400

Peak T (tryptophan-like)

Fulvic acid-like 400

450

500

550

Em (nm)

265

Figure 3. (a) A rough division of excitation-emission wavelength regions according to FQ

266

distribution of the hydrophobic/hydrophilic components, in comparison with (b) classic

267

division of the EEM map based on chemical species 16,17.

268 269

3.3.

Quasi-quantitative

Relationship

270

Hydrophobic/Hydrophilic Contents

between

Fluorescence

and

271

Since different hydrophobic/hydrophilic components present different characteristic EEM

272

regions, the fluorescence profile of the total DOM (consisting of the different components)

273

may vary as a function of the relative contents of the components. To this end, it was further

274

explored whether there was any significant numerical correlation between the total DOM’s

275

fluorescent properties and its hydrophobic/hydrophilic composition. Pearson’s correlation

276

between the regional fluorescence proportion (fi) and the hydrophobic/hydrophilic contents

277

(TOC proportions) was scanned over different wavelength regions (Exi ± ∆λ, Emi ± ∆λ),

278

using the technique described in Section 2.5. For each ∆λ, the best correlation coefficient (i.e.

279

with maximum |ri|) and its corresponding Ex/Em region are shown in Figure 4a-c.

15



280

For HIS (Figure 4a), the best significant positive ri occurred in a sufficiently large region

281

of (Ex, Em) = (200-400, 300-550) nm which was regarded as a robust region for the positive

282

correlation between fi and HIS proportion with ri = 0.78 (p < 0.01). Similarly, Em < 300 nm

283

was regarded as a robust region for negative correlation between fi and HIS proportion with ri

284

= -0.71 (p < 0.05). The negative correlation must be due to the influence of non-HIS fractions

285

on the relative fluorescence in the corresponding region. For HOA (Figure 4b), the best

286

positive correlation region was (Ex, Em) = (200-285, 340-465) nm with a very high ri of 0.94

287

(p < 0.0001), and the best negative correlation region appeared at Ex > 270 nm and Em < 400

288

nm with ri = -0.68 (p < 0.05). For HOB (Figure 4c), significant positive correlations only

289

occur in relatively narrow regions with small ∆λs.

290

The best positive/negative correlation regions for HIS, HOA and HOB are summarized in

291

Figure 4d. The ri and confidence of positive correlation followed the order HOA > HIS >

292

HOB. The positive correlation regions are generally consistent with the FQ-based division of

293

characteristic regions of the individual components as depicted in Figure 3a. This indicates

294

that the regional fluorescence of the total DOM retains the fluorescent features of the

295

individual components, and may be quantitatively related to the relative contents of the

296

components.

297

16


Page 16 of 27

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298

300

Ex range

200

0.8 0.6 0.4 0.2 0.0

Negative r (significant) 500

Negative r (insignificant)

400 300

-0.2 -0.4 -0.6

Positive r (significant)

500 Em range

400 300

Ex range

200

200

0.4 0.2 Negative r (insignificant)

-0.2 -0.4 -0.6 -0.8 -1.0

200 0

20

40

60

80

100

120

140

0

20

40

60

∆λ (nm)

400 300

Positive r (insignificant)

0.6 0.4 0.2

Ex range 200

0.0 Negative r (insignificant)

500

-0.2 -0.4

400

-0.6 -0.8

300

-1.0

200 0

20

40

60

80

140

450

0.8

Em range

Negative r (significant)

120

1.0

100

120

400

HIS (r = +0.78, p = 0.0077)

350

Ex (nm)

Optimal region


500

100

(d) Optimal regions for correlation as scanned

Correlation coefficient

600

80

∆λ (nm)

(c) DOM fluorescence vs. HOB proportion

Ex or Em (nm)

0.6

0.0

500

300

-1.0

1.0 0.8

Negative r (significant)

400

-0.8


Optimal region

400

600 1.0

Optimal region

Optimal region

Em range

Optimal region

Ex or Em (nm)

500


Ex or Em (nm)



600

(b) DOM fluorescence vs. HOA proportion


(a) DOM fluorescence vs. HIS proportion

HOA (r = -0.68, p = 0.029)

300

250

200 250

140

HOA (r = +0.94, p = 6.8e-5)

HIS (r = -0.71, p = 0.022) HOB (r = +0.68, p = 0.032)

300

∆λ (nm)

350

400

450

500

550

Em (nm)

299 300

Figure 4. Scanning the optimal EEM regions for correlation of relative fluorescence with (a)

301

HIS proportion, (b) HOA proportion, and (c) HOB proportion, with the optimal regions

302

summarized in (d). The scanning process is illustrated in Figure S2. ∆λ denotes the half-width

303

of the scanning window.

304 305

Linear regression was further performed to assess the quantitative relationships between

306

regional fluorescence proportion (fi) and hydrophobic/hydrophilic contents in the total DOM.

307

Figure 5a shows the regression of HOA proportion as a function of fi in the region of (Ex, Em)

308

= (200-285, 340-465) nm at 95% confidence intervals. The regression model was highly

309

significant (F-test, p < 0.0001) with R2 = 0.876 and a high leave-one-out cross-validation Q2 17



310

= 0.827. Monte Carlo simulation (inset of Figure 5a) shows that the simulated R2 surpassed

311

0.80 for over 95% of the cases, indicating that the model might be reliable for potential

312

quasi-quantitative prediction of HOA content in the total DOM. As fi varies from 0.35 to 0.45,

313

HOA’s proportion varies from 0.2 to 0.4 which covers the general range as reported for

314

full-scale MBRs treating municipal wastewater 38,39. Figure 5b presents the regression of HIS

315

proportion as a function of fi in the region of (Ex, Em) = (200-400, 300-550) nm, with R2 =

316

0.609 (p < 0.01) and Q2 = 0.395. Monte Carlo simulation (inset of Figure 5b) shows that the

317

simulated R2 is widely distributed between 0 and 1, and is smaller than 0.5 in 30% of the

318

cases, indicating that the regression model was less robust. The wide confidence intervals

319

indicate that the HIS content is difficult to be accurately predicted using the present model.

320

Hypothetically, this might be related to the relatively high non-fluorescence portion in HIS

321

(as inferred from the relatively low overall fluorescence density of HIS, see Section 3.1).

322

Nonetheless, this non-fluorescence portion of HIS would bring minimal interference to the

323

significance of the relationship betwen fi and HOA content, as assessed in Figure S4. For

324

further exploration of any possible quasi-quantitative relationships to predict the HIS content,

325

HIS might be subdivided into HIA/HIB/HIN in future studies.

326

18


Page 18 of 27

Page 19 of 27


327

0.33 0.31 0.29 0.27

0.7

y = 1.621x − 0.356 R2 = 0.876 Q2 = 0.827

0.25 0.23 0.21 0.19 0.17

15 12 9 6 3 0 0.6 0.7

Simulated R2

1

0.9 0.8 0.7

0.6

0.6

0.55

0.5

0.5 0.45 0.4 0.35 y = 4.18x − 4.14 R2 = 0.609 Q2 = 0.395

0.3 0.8 0.9

0.15 0.35 0.36 0.37 0.38 0.39 0.4 0.41 0.42 0.43 0.44

328

0.65

1

Experimental Fitted line 95% C.I. mean 95% C.I. individual

0.25 0.2 0.93

0.94

0.95

0.96

Frequency (%)

TOC proportion of HOA

0.35

0.75

TOC proportion of HIS

0.37

0.8

Experimental Fitted line 95% C.I. mean 95% C.I. individual

Frequency (%)

0.39

(b) HIS

5 4

0.4

3 2

0.3 0.2

1 0

0 0.2 0.4 0.6 0.8

0.1

1

Simulated R2

0.97

0.98

0.99

Probability of fitted line track by Monte Carlo simulation

(a) HOA

1

0

Regional fluorescence proportion

Regional fluorescence proportion

329

Figure 5. Regression analysis of (a) the dependence of HOA proportion on the regional

330

fluorescence proportion of (Ex, Em) = (200-285, 340-465) nm, and (b) the dependence of

331

HIS proportion on the regional fluorescence proportion of (Ex, Em) = (200-400, 300-550) nm.

332

R2 and the leave-one-out cross-validation Q2 are marked beside the fitting lines. The insets

333

are results of 10,000-time Monte Carlo simulation based on t-distribution of the mean-square

334

errors of the models.

335 336

3.4. Implication

337

The DOM samples from the 10 WWTPs have revealed statistically universal trends in the

338

relationships between EEM fluorescent properties and hydrophobic/hydrophilic contents.

339

Qualitatively, characteristic EEM wavelength regions for the hydrophobic/hydrophilic

340

components of DOM were identified on the basis of FQ. Quantitatively, significant Pearson’s

341

correlations were detected between some of the regional fluorescence proportions (fi) and the

342

hydrophobic/hydrophilic contents. Particularly, a linear regression model was further

343

established describing the numerical dependence of HOA proportion on fi. These 19



344

relationships may provide clues for qualitative indication of the overall DOM hydrophobicity

345

level, or quasi-quantitative detection of HOA content changes in MBR systems, and hence

346

provide a foundation for further research and development on EEM-based monitoring of

347

hydrophobic/hydrophilic compositon. With the popularization of fluorescence-based on-line

348

detection techniques in the field of water/wastewater treatment 14,17,40-43, real-time monitoring

349

of the hydrophobic/hydrophilic contents of DOM should be quite practicable and promising.

350

For practical application of the findings of this study, it would be worthwhile to further track

351

the dynamic performance of the fluorescence indicators during long-term operation of MBR.

352

The EEM-monitored DOM hydrophobicity may be a useful complement to the existing

353

on-line monitoring terms (e.g. suspended solids, effluent quality, flux, trans-membrane

354

pressure and temperature) to provide timely grasp of the process status. In MBRs, it could be

355

used for early warning of status change that is possibly related to pollutant removal, effluent

356

quality and membrane fouling. To fully exploit the potential of fluorescence-based

357

monitoring of DOM hydrophobicity, further study needs to be devoted to the linkages among

358

DOM hydrophobicity, fluorescent properties, and process status/functions.

359 360

ASSOCIATED CONTENT

361

Supporting Information

362

Figures showing procedure for hydrophobic/hydrophilic fractionation, scheme of scanning

363

EEM regions for fluorescence correlation with hydrophobic/hydrophilic proportions,

364

contours of FQ between different hydrophobic/hydrophilic components, and regression of the

365

relationship between the regional fluorescence proportion and the HOA proportion (corrected by 20


Page 20 of 27

Page 21 of 27


366

excluding the non-fluorescence portion of HIS from the total solution). Tables showing basic

367

information of the 10 WWTPs for DOM sampling, properties of the influent, effluent and

368

mixed liquor at the sampling time, and contents of polysaccharides, proteins and humics in

369

HIS, HOA and HOB.

370 371

AUTHOR INFORMATION

372

Corresponding Authors

373

*E-mails: [email protected] (Y. Shen); [email protected] (X. Huang).

374

ORCID

375

Kang Xiao: 0000-0001-6475-1960

376

Shuai Liang: 0000-0002-4349-2792

377

Peng Liang: 0000-0001-7345-0844

378

Xia Huang: 0000-0003-4076-1464

379

Notes

380

The authors declare no competing financial interest.

381 382

ACKNOWLEDGMENTS

383

This work was supported by the National Natural Science Foundation of China (No.

384

51778599, No. 21407147). We gratefully thank the workers and engineers of the WWTPs for

385

their kind help with the sampling.

386 387

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