Polarity and Molecular Weight of Compost-Derived Humic Acids

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Agricultural and Environmental Chemistry

Polarity and molecular weight of compost-derived humic acids impact bio-dechlorination of pentachlorophenol Ying Yuan, Beidou Xi, Xiao-Song He, Wenbing Tan, Hui Zhang, Dan Li, Chao Yang, and Xinyu Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05864 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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Journal of Agricultural and Food Chemistry

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Polarity and molecular weight of compost-derived humic

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acids impact bio-dechlorination of pentachlorophenol

3

Ying Yuan a, b, Beidou Xi a, Xiao-Song Hea*, Wenbing Tan a, Hui Zhang a, Dan

4

Li a, Chao Yang a, Xinyu Zhao a

5

a

6

Research Academy of Environmental Sciences, Beijing 100012, China

7

b

8

and Pollution Control, Tsinghua University, Beijing 100084, China

State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese

School of Environment and State Key Joint Laboratory of Environment Simulation

9 10 11 12 13 14 15 16 17 18 19

Corresponding author at: No.8 Dayangfang, Beiyuan Road, Chaoyang district,

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Beijing 100012, China. Tel.: +86 10 84915307, +86 10 18800198488; fax: +86 10

21

84913133

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E-mail address: [email protected] (X.-S. He)

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ACS Paragon Plus Environment


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Abstract: Compost-derived humic acid (HAs) as cheap soil conditioners have

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potential to facilitate pentachlorophenol (PCP) bio-dechlorination but less of proofs

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and studies. To clarify this, PCP bio-dechlorination mediated by compost-derived

4

HAs under Fe(III) reduction condition was investigated. The reverse phase

5

high-performance liquid chromatography and high-performance size exclusion

6

chromatography were employed to identify the functional components within

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compost-derived HAs. Our results showed that compost-derived HAs facilitated the

8

bio-dechlorination of PCP under Fe(III) reduction condition, and 4 kinds of

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by-products were detected during the process. The relatively hydrophilic and high

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molecular weight (MW) components within compost-derived HAs presented

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significant associations with the concentration of by-products from bio-dechlorination

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of PCP in Fe2O3 reduction condition. On the contrary, the hydrophobic and low MW

13

components were the main functional components for PCP bio-dechlorination in

14

Fe3O4 reduction environment. These findings clarified the effects of polarity and MW

15

of compost-derived HAs on PCP bio-dechlorination, giving clue to optimize

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composting technology to utilize compost products in in-situ contamination

17

remediation of paddy soil.

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Keywords: Pentachlorophenol bio-dechlorination; Composting; Humic acids;

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High-performance liquid chromatography; Fe(III) oxides reduction

2


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INTRODUCTION

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Pentachlorophenol (PCP) is one of the most significant and frequently detected

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chlorinated organic pollutants in environment

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extensive contamination of soil, surface and groundwater, where it can be toxic to

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plants, animals and humans 4. PCP is redox-active, and the bioremediation of PCP in

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paddy soil is always research hotspot

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bioremediation of PCP have verified that microbial reductive dechlorination is an

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eco-friendly and cost-competitive alternative to purify the soil environment for the

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transformation of the chlorinated compounds under anoxic conditions 6. However,

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anaerobic bio-dechlorination of PCP always suffers from low transformation rate due

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to the low content of electron donators such as nutrients and electron shuttles such as

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humic substance in natural environment

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shuttles had more effects on the bio-dechlorination of PCP 5. Therefore, the

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supplement of electron shuttles in PCP-contaminated environment would significantly

34

improve its bio-dechlorination rate in long period 9.

7,8.

1-3.

5.

Its bioaccumulation has led to the

Previous studies concerning the

Compared with the nutrients, electron

10,11.

35

Composting has been widely used in the disposal of the organic wastes

36

Compost products contain many kinds of organic matters such as humic acids and

37

fulvic acids. These cheap organic matters have been confirmed to be able to improve

38

the composition and nutrient content of soil. Recently, redox properties of

39

compost-derived organic matters gradually raised concerns

40

organic matters have been confirmed to facilitate the reduction of Fe(III) to Fe(II)

41

and nitrobenzene to aniline

14

12-14.

Compost-derived 13

under anaerobic condition in our previous studies. 3



42

Among

43

compost-derived HAs had longer effects on soil environment due to their stable

44

structures

45

the electron transfer between extracellular respiration bacteria and iron (III)

46

Meanwhile, the Fe(III) reduction condition was considered to be the key factor

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driving bio-dechlorination of PCP in natural anaerobic environment 18. Therefore, we

48

speculated

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bio-dechlorination of PCP in Fe(III) reduction environment. However, compared with

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natural HAs, compost-derived HAs have significantly different organic carbon

51

precursors and shorter transformations, resulting in the different redox properties

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between

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compost-derived HAs like natural HAs would act as electron shuttles to facilitate the

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PCP transformation was ambiguous. The main factors and functional components in

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compost-derived HAs impacting on bio-dechlorination of PCP also remained unclear.

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These shortcomings knowledge seriously hindered the application of compost

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products in contamination remediation, further limited the innovation of

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contamination remediation technology especially for the low-cost technologies.

59

the

15.

redox-active

organic

matters

existed

in

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compost

products,

Compost-derived HAs were able to act as electron shuttles to facilitate

that

compost-derived

compost-derived

HAs

HAs

and

have

natural

potential

HAs

13.

to

13,16,17.

facilitate

Therefore,

the

whether

Previous researches showed that electron transfer mediated by electron shuttles 16.

60

was most effective in aqueous solution through contact mechanism

61

between the Fe(III) reduction bacteria (electron donors) and the HAs (electron

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shuttles) occurred mainly on the cell membranes of the Fe(III) reduction bacteria,

63

because the cell membranes have special reaction sites for the HAs 4


The contact

19,20.

Cell

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64

membranes were polar, therefore, the polarity of the HAs (electron shuttle) would

65

impact the electron transfer between the Fe(III) reduction bacteria and the HAs

66

Moreover, the reaction sites in cell membranes also have limited size range 21, which

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would result in the differences of electron transfer rate between humic-like

68

components with different molecular weight (MW). Therefore, we hypothesized that

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the polarity and MW of compost-derived HAs would have significant effects on PCP

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bio-dechlorination. Unfortunately, no direct proof demonstrated it so far. To clarify

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these effects, reverse phase high-performance liquid chromatography (RP-HPLC) and

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high-performance size exclusion chromatography (HPSEC) were employed to reveal

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the polarity and MW of compost-derived HAs, respectively 22. Moreover, lactate and

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PCP were selected as the electron donor and acceptor, respectively. Fe2O3 and Fe3O4,

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two common Fe(III) oxides in natural soil environment, were also involved, due to the

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bio-dechlorination of PCP was always coupled with iron (III) reduction 1. Moreover, S.

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oneidensis MR-1, a kind of facultative extracellular respiration bacteria, was used as

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iron-reducing bacterium in this work, given the facultative anaerobe condition was

79

widely existed in natural environment.

20.

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Therefore, the objectives of this work were to verify the ability of

81

compost-derived HAs to facilitate the bio-dechlorination of PCP, to clarify the effects

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of polarity and MW of compost-derived HAs on PCP bio-dechlorination during the

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reduction of Fe(III) oxides by iron-reducing bacterium, and to identify the effective

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components within compost-derived HAs responsible for PCP bio-dechlorination.

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MATERIALS AND METHODS 5



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Composting process and sample collection. Composting continued for 47 days

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in an indoor composting reactor with a volume of 34 L and diameter × height of 330

88

mm × 400 mm (Shizuoka co., Ltd, Japan). Composting materials consisted of kitchen

89

wastes (10.5 kg, from canteen), soil (9 kg), sawdust (0.23 kg) and a composite

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microbial system (1.6 kg). The ventilation was controlled at 0.5 L·min-1·kg-1 during

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the composting. The changes of temperature and pH during composting were shown

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in Fig. S1. Compost samples were collected after 0, 3, 6, 8, 13, 19, 35 and 47 d of

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composting in the depth of 5, 15 and 25 cm, respectively. All samples were

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immediately freeze-dried (FD-1A-50, Liwen, China) and stored in a -20

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HAs extraction. Compost-derived HAs was obtained according to the IHSS standard

96

assay (See Supporting materials).

freezer for

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Analytical methods. The dissolved organic carbon (DOC) of all samples was

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measured by a TOC automatic analyzer (MultinN/C2100TOC/TN) after being filtered

99

by 0.45 I

mixed cellulose ester filter membranes (Weining, China). Specific UV

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absorbance values SUVA254 (=UV254×100/DOC)

101

24

102

corresponding DOC concentration.

23

and SUVA290 (UV290×100/DOC)

were calculated by dividing the absorbances at 254 nm and 290 nm by the

103

RP-HPLC and HPSEC chromatogram. RP-HPLC and HPSEC analyses were

104

conducted by Agilent 1100 LC systems (Agilent, CA, USA) equipped with a diode

105

array detector and a fluorescence detector. For the RP-HPLC analyses, an Eclipse

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XDB-C18 (150 mm × 4.6 mm, 5 I ; column (Agilent, CA, USA) was applied, and a

107

mixture of acetonitrile (5%) and ammonium acetate (10 mM, 95%) were employed as 6


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108

the mobile phase. Chromatography was performed with the flow velocity of 1

109

mL·min-1 at 30 °C, and the injection volume was 100 I&

110

application, a MIXED-M (300 mm × 7.5 mm, *I ; column (Agilent, CA, USA) was

111

used, and the chromatography parameters were the same to that in RP-HPLC analyses.

112

The RP-HPLC and HPSEC diode array detector chose 254 nm and 290 nm. The

113

fluorescence detector used Ex/Em = 270/475 nm and Ex/Em = 375/440 nm scans to

114

analyze

115

excitation–emission matrix (3DEEM) fluorescence spectra 12,13.

compost-derived

HAs

on

the

basis

of

the

25.

For the HPSEC

three-dimensional

116

Reduction of Fe(III) oxides experiment. The reduction of Fe(III) oxides

117

experiments were conducted in a 100 mL brown anaerobic bottle (Heklas, China).

118

First, 20 mL compost-derived HA (~50 mg·L-1, as electron shuttle) was injected into

119

the anaerobic bottle, then Fe(III) oxides (Fe2O3 or Fe3O4 obtained from Sinopharm

120

Chemical ReagentCo., Ltd) and sodium lactate (Chinese medicine group) were

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injected into the anaerobic bottles with the initial concentration were 1 mM·L-1 and 5

122

mM·L-1, respectively. Phosphate buffer (20 mL, 0.2 M, pH=7) was instead of

123

compost-derived HAs in control experiment. Second, 20 mL MR-1 cell suspension

124

[(1-5)·107 CFU mL-1] in LM-lactate (LML) medium 26 was injected into the anaerobic

125

bottle, then purged with 100% N2 for 20 min and immediately stoppered with butyl

126

rubber bungs. All anaerobic bottles were static incubation in an anoxic glovebox (N2,

127

atmosphere at 25±1 , O2 H > I > J > K > L. Meanwhile, 5 kinds of humic-like components (the

298

hydrophility of them were M> N > O > P > Q) with the fluorescence wavelengths of

299

Ex/Em=375/440 nm were also eluted (Fig. 5).

13

combined with RP-HPLC were used to

300

Among the 12 kinds of humin-like fluorescence components identified by the

301

RP-HPLC, the relatively hydrophilic components showed better correlations with the 17



302

concentrations of PCP and its by-products than the hydrophobic counterparts in Fe2O3

303

reduction environment (Tables S4 and S5). Meanwhile, the Fe2O3 T-ETCs of

304

compost-derived HAs also showed significant correlations with these hydrophilic

305

components within compost-derived HAs (Table S6). These results demonstrated that

306

the polarity of compost-derived HAs had significant effects on the transformation of

307

PCP during Fe(III) oxides reduction mediated by iron-reducing bacterium. It further

308

suggested that the relative hydrophilic components within compost-derived HAs

309

could effectively transfer electron between the iron-reducing bacterium, Fe2O3 and

310

PCP. One possible reason responsible for the results was that the relative hydrophilic

311

components within compost-derived HAs had a higher concentration in aqueous

312

solution system than the hydrophobic ones. Therefore the hydrophilic components

313

could complex with Fe(II) more easily and get a better PCP transformation than the

314

hydrophobic ones

315

compost-derived would facilitate the release of the PCP absorbed on Fe(III) oxides,

316

which would enhance the transformation of PCP in our systems 18, 33-35.

6,18.

Another reason was that hydrophilic components within

18


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8

K

1.2 J

0.9 H 0.6

L

0.3 0.0 0

2

317

4 6 Retention time (min)

8

10

2


8

2 1

Fluorescence intensity

3

6 4 2 0

0 0

2


8

10

2


8

2


8

1

0

35d

2


8

10

47d

6

7 6 5 4 3 2 1

10

2

10

5 4 3 2 1 0

0 0

3

7

8

8

4

0

0

19d

4

2

10

10

5

4

0 0



0.3

13d

6

319

0.6

0.0

7

318

0.9

8d

6


1.5


1.2


FG

5

6d

3d



0d

I

1.8

0

2


8

10

0

2


Fig. 4. RP-HPLC time-maps of the 8 compost-derived HA samples at 270 nm excitation and 475 nm emission wavelengths.

19


8

10


0d

2.1

6

3d

4 3 N M O

2

Q

1


5 1.8 1.5 1.2 0.9 0.6

0 8

0

2


6 5 4 3 2 1

8

4 3 2 1 2


8

3.0

4 3 2 1

35d

0

2


8

10

0

2


8

2.0 1.5 1.0

1

2


8

10

47d

2.0 1.5 1.0 0.5

0

10

2

2.5

2.5

0

3

0

10

0.5

0

4

0 0

10

19d

5



10

13d

7

322

5


320



2

8d

5

0 0

321

6d


P



6

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2


8

10

0

2


Fig. 5. RP-HPLC time-maps of the 8 compost-derived HA samples at 375 nm excitation and 440nm emission wavelengths.

20


8

10

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323

Among the 3 kinds of the components identified at UV 254 nm in RP-HPLC, the

324

most hydrophobic one presented a significant negative correlation with the

325

concentration of the 2,4-dichlorophenol in Fe2O3 reduction environment (Table S7).

326

This finding indicated that, the hydrophobic components might hinder the electrons

327

transfer between the iron-reducing bacterium, Fe2O3 and PCP, whereas the relatively

328

hydrophilic aromatic components within compost-derived were the effective ones for

329

PCP bio-dechlorination in Fe2O3 reduction environment. In addition, the SUVA254 of

330

the components within compost-derive HAs identified by RP-HPLC also showed a

331

significant correlation with the T-ETCs of compost-derived HAs (Table S6). This

332

finding indicated that the aromatic structures existed in compost-derived HAs could

333

effectively facilitate the electron transfer between the iron-reducing bacterium and

334

Fe(III) oxides, and this function of compost-derived HAs was similar to natural HAs

335

27.

336

ones were effective for the PCP bio-dechloriantion during Fe(III) oxides reduction.

Among these components identified by RP-HPLC, only the relatively hydrophilic

337

Effect of the molecular weight of compost-derived HAs on the

338

transformation of PCP. Molecular weight (MW) of humic like substances have been

339

confirmed to influence the ETC of HAs

340

compost-derived HAs would had effects on the bio-dechlorination of PCP during

341

Fe(III) oxides reduction. In order to clarify the possible effects, high-performance size

342

exclusion chromatography (HPSEC) was used to reveal the MW characterization of

343

compost-derived HAs. Based on HPSEC, three kinds of aromatic components (S-T)

344

were identified at 254 nm using UV detector. Meanwhile, three kinds of quinone

32,33,36,

so we speculated that the MW of

21



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structures (U-W) within compost-derived HAs were identified at 290 nm as well (Fig.

346

6). The MW order of the aromatic components was S > R > T, and that of quinone

347

structures was U > V > W due to the higher MW components with shorter retention

348

time in HPSEC. UV absorptions of compost-derived HAs at 254 and 290 nm were

349

also similar to each other in HPSEC, confirming that the aromatic components within

350

compost-derived HAs contained quinone groups.

160

(a)

120 80 40

0d 3d 6d 8d 13d 19d 35d 47d S

R

UV absorption value at 290 nm

UV absorption value at 254 nm

345

T

0 0

3 6 9 Retention time (min)

140 120

(b)

100 80 60 40 20

V

W

U

0 0

12

0d 3d 6d 8d 13d 19d 35d 47d

2 4 6 Retention time (min)

8

10

Fig. 6. UV absorption value of compost-derived HAs in HPSEC: (a) UV absorption value of compost-derived HAs at 254 nm in HPSEC; (b) UV absorption value of compost-derived HAs at 290 nm in HPSEC. 351

Based on 3DEEM combined with HPSEC, three kinds of humic-like components

352

(X-Z) with the Ex/Em wavelength of 270/475 nm (Fig. S4) and three kinds of

353

humic-like components (AA-AC) with the Ex/Em wavelengths of 375/440 nm within

354

compost-derived HAs were also identified (Fig. S5). Among the six humic-like

355

components, the low MW humic-like components showed significant negative

356

correlations with the concentration of 2,6-dichlorophenol in Fe2O3 reduction

357

environment and significant positive correlations with the concentration of PCP in

358

Fe3O4 reduction environment (P

Polarity and Molecular Weight of Compost-Derived Humic Acids

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