Coupling between Pentachlorophenol Dechlorination and Soil Redox

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Coupling between pentachlorophenol dechlorination and soil redox as revealed by stable carbon isotope, microbial community structure, and biogeochemical data Yan Xu, Yan He, Qian Zhang, Jian-Ming Xu, and David E. Crowley Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es505040c • Publication Date (Web): 08 Apr 2015 Downloaded from http://pubs.acs.org on April 14, 2015

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Environmental Science & Technology

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Coupling between pentachlorophenol dechlorination and soil redox

2

as revealed by stable carbon isotope, microbial community structure,

3

and biogeochemical data Yan Xu† Yan He †, ‡,∗∗ Qian Zhang† Jianming Xu†,∗∗ David Crowley‡

4 5

†

6

Provincial Key Laboratory of Subtropical Soil and Plant Nutrition, Zhejiang

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University, Hangzhou 310058, China.

8

‡

9

Riverside, CA 92521, USA.

Institute of Soil and Water Resources and Environmental Science, Zhejiang

Department of Environmental Sciences, University of California, Riverside,

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11 12

Abstract Art

1

ACS Paragon Plus Environment


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Abstract

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Carbon isotopic analysis and molecular-based methods were used in conjunction

15

with geochemical data sets to assess the dechlorination of pentachlorophenol (PCP)

16

when coupled to biogeochemical processes in a mangrove soil having no prior history

17

of anthropogenic contamination. The PCP underwent 96% dechlorination in soil

18

amended with acetate, compared to 21% dehalogenation in control soil. Carbon

19

isotope analysis of residual PCP demonstrated an obvious enrichment of

20

3.01±0.1%). Molecular and statistical analyses demonstrated that PCP dechlorination

21

and Fe(III) reduction were synergistically combined electron-accepting processes.

22

Microbial community analysis further suggested that enhanced dechlorination of PCP

23

during Fe(III) reduction was mediated by members of the multifunctional family of

24

Geobacteraceae. In contrast, PCP significantly suppressed the growth of SO42-

25

reducers, which, in turn, facilitated the production of CH4 by diversion of electrons

26

from SO42- reduction to methanogenesis. The integrated data regarding stoichiometric

27

alterations in this study gives direct evidence showing PCP, Fe(III) and SO42-

28

reduction, and CH4 production are coupled microbial processes during changes in soil

29

redox.

13

C (εC, -

2


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INTRODUCTION

31

Biogeochemical cycles involving carbon (C), nitrogen (N), sulfur (S) and iron

32

(Fe) are driven by fundamental oxidation-reduction processes in which electrons flow

33

from carbon substrates and other electron donors to those elements that serve as

34

terminal electron acceptors for microbial respiration1. These same active redox

35

processes also play a key role in the transformation of biogenic minerals and catalyze

36

the oxidation and reduction of anthropogenic chemicals, including many organic

37

contaminants (OCs)2. The speciation, bioavailability and toxicity of the OCs

38

determine the type and magnitude of these biotransformations and select for different

39

degrader communities that can influence contaminant dynamics under specific redox

40

conditions3.

41

Pentachlorophenol (PCP) is a highly toxic, mutagenic and carcinogenic

42

polychlorinated organic compound that is widely used as a wood preservative and

43

versatile insecticide4-5. Under anaerobic conditions, reductive dechlorination is

44

thought to be coupled to biogeochemical processes driven by electrons flow from

45

hydrogen, reduced minerals, and carbon substrates (e.g. acetate, lactate) to PCP,

46

which serves as an electron acceptor6-7. Usually, reducing reactions of various ionic

47

species in soils such as NO3-, Fe(III), Mn(IV) and SO42- that serve as terminal electron

48

acceptors which are active and important redox reactions in anaerobic environments8.

49

Thus, the dechlorination of PCP depends on the abundance and activity of

50

dehalorespiring bacteria which could be influenced by interaction with alternative

51

terminal electron accepting processes. In a previous study using a liquid pure culture

52

system, we demonstrated that the iron-reducing bacterium, Clostridium beijerinckii Z,

53

could dechlorinate PCP under anaerobic conditions, and that the dechlorination rate

54

was accelerated during simultaneous Fe(III) reduction6. This gave preliminary

55

evidence that the anaerobic declorination of PCP was interactively affected by

56

coexisting electron acceptors such as Fe(III). As natural soil systems contain various

57

and abundant electron donors/acceptors, anaerobic dechlorination of PCP is expected

58

to be more complex. Therefore, anaerobic transformation of PCP still needs to be

59

examined by qualitative and quantitative data to reveal the relevant functional

60

microbial groups, and interactions between PCP and multiple environmental variables

61

that control redox reactions in soils.

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Compound specific isotope analysis (CSIA) of stable carbon (C) has been

63

increasingly used to identify and quantify the degradation parent OCs in contaminated

64

systems9-10. The method is based on isotopic fractionation (13C/12C) that occurs during

65

preferential transformation of molecules containing lighter isotopes (12C) relative to

66

those containing heavier isotopes (13C) in the target molecule. In this manner, the

67

residual parent compound becomes progressively enriched in heavy isotopes (13C) and

68

depleted in light isotopes (12C) during the degradation of OCs11. When there are also

69

non-destructive processes such as dilution, sorption, and volatilization that lead to

70

disappearance of the target compound, the CISA method provides a means to estimate

71

the contribution of enzymatic processes12. Recently, this method has been used in

72

studies of anaerobic transformation of chloroethylenes (CEs)11-15 and chlorophenols16,

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and for studies on the aerobic degradation of aromatic compounds or methyl-tert-

74

butyl ether (MtBE)17-18. Additionally, when used in combination with culture-

75

independent molecular techniques and stoichiometric analyses of environmental

76

variables, CSIA may provide further insight into the ‘black box’ of microorganisms

77

that are involved in biotic transformation processes, and lead to a better understanding

78

of the mechanisms that underpin biogeochemical transformations of pollutants in

79

soils19.

80

In this study, soil incubation experiments were carried out to examine the

81

processes that are involved in reductive dechlorination of PCP when coupled with

82

biogeochemical redox cycles for C, Fe and S. Analytical procedures were developed

83

for: (1) stable carbon isotope analysis to assess the extent and mechanisms involved in

84

the reductive dechlorination of PCP, (2) correlation analysis between PCP

85

dechlorination and measurement of relevant environmental factors (chloride ion (Cl-),

86

dissolved organic carbon (DOC), dissolved organic nitrogen (DON), total HCl-

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extractable Fe (FeHCl), redox potential (Eh), carbon dioxide (CO2), ferrous ion (Fe(II),

88

bivalent manganese (Mn(II), sulfate (SO42-), sulfides (RSCs), methane (CH4)) during

89

incubation, and (3) use of phospholipid fatty acid (PLFA) profiles, quantitative real-

90

time polymerase chain reaction (qPCR) and high-throughput sequencing (MiSeq) to

91

assess microbial community structures and specific functional genes that are relevant

92

to reductive dechlorination of PCP. Our hypothesis is that the reductive dechlorination

93

of PCP and the natural biogeochemical soil redox (reduction of Fe(III) and SO42-, and

94

emission of CH4) were all mutually-influenced coupling processes. 4


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MATERIALS AND METHODS

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Soils

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Soil samples were collected from a coastal mangrove soil near Taishan city in

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Guangdong province in China (21°48.991’N, 112°27.848’E). The soil had a long

99

history of strongly reducing conditions and contained abundant reduced iron minerals

100

and sulfides, but was deficient in nitrogen. The soil samples were collected at 20 cm

101

intervals down to a 1 meter depth in the soil profile, after which the soils were air-

102

dried naturally and passed through a 1 mm sieve prior to use in experiments. The

103

basic physicochemical properties of the five soil layers were analyzed and the results

104

are described in Section S-1 and Figure S1 in the Supporting Information (SI).

105

Soil Anaerobic Incubation

106

The sieved soils were divided into two equal portions for generation of PCP

107

spiked and unspiked treatment groups. To prepare PCP spiked soils, 0.3 kg portions of

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soil were spiked with 10 mL PCP solution (3000 mg L-1 dissolved in methanol),

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mixed evenly using a glass rod, and dried for 24 h to remove the methanol solvent.

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The spiked soil portion from each profile was then added to 0.3 kg of the

111

corresponding soil four more times to provide an initial concentration of 20 mg PCP

112

kg-1 soil. The polluted soils were then allowed to equilibrate for one week at room

113

temperature. All of the mixing processes were performed using an anaerobic chamber

114

(Don Whitley Scientific, England) to exclude air and prevent oxidation of the soil

115

mineral components.

116

The effects of acetate and PCP were compared using four treatments using 1:1

117

(w/v) soil/water mixtures prepared from each soil layer. In brief, mixtures containing

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15 g soil in 15 mL H2O were incubated in 150 mL serum bottles containing: (i)

119

acetate + PCP spiked soil, (ii) PCP spiked soil, (iii) acetate + unspiked soil, and (iv)

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unspiked, unamended soil, which served as a control treatment. An abiotic soil

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treatment was also included, using soil samples that were sterilized by γ-irradiation at

122

50 kGy. The sterile water was purged of oxygen by flowing N2 through the water for

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5 hr at a gas flow rate of 5 L min-1. Acetate was added to a concentration of 20 mmol

124

kg-1 soil, and the bottles were likewise purged with N2 (99.99%) for 10 min (5 L min-1)

125

using a 10 mm diameter rubber hose, after which the bottles were sealed with Teflon-

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coated butyl rubber stoppers and crimp seals. The purging time (10 min) and flowing 5



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rate (5 L min-1) were selected based on a preliminary study, which testified to a

128

satisfactory elimination of O2 from the experimental systems. Both treatments were

129

incubated at 30 oC in the anaerobic chamber under a N2 stream. Triplicate samples

130

from each treatment were destructively sampled after 7, 14, 21, 28, 42, 63 and 84 days

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of incubation.

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Analytical Methods

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Chemical and Microbial Analytical Methods

Microbial communities and

134

functional groups, residual PCP and its metabolites in soils, as well as other major

135

molecules and ions of environmental variables were measured following previously

136

described methods that are summarized in Section S-2 and Table S1 4,20.

137 138

139

Carbon Stable Isotope Analysis

The carbon isotope fractionation in PCP

transformation was expressed according to Rayleigh equation11: 1000 + δ 13Ct C ε ln( )= ln t 13 1000 + δ C0 1000 C0

(1)

140

where δ13Ct and δ13C0 are the carbon isotope signatures of the given substrate at time t

141

and the initial time, respectively; Ct and C0 are the substrate concentration at time t

142

and the initial time; ε is the isotope enrichment factor. See detailed analytical methods

143

in Section S-2.

144

Data Processing and Statistical Analysis

Multivariate statistical analyses

145

were carried out using R and SPSS software. Relevant half reactions of electron

146

acceptors during incubations of 84 days were used to calculate the transferred electron

147

equivalent (eeq). Pearson Correlation coefficients, Multiple Correspondence Analysis

148

(MCA) and RV correlations were used to analyze the dynamic processes of

149

environmental parameters and the relationships between environmental variables and

150

microbial community structures (MCS).

151 152

RESULTS

153

PCP Dechlorination and Proposed Pathway under Anaerobic Condition

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The extent of PCP transformation and formation of intermediate products in soils

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with or without acetate addition are shown in Figure 1. The PCP dechlorination

156

extents in each soil layer were significantly greater following acetate addition than

157

those without acetate addition. In the treatment without acetate (Figure 1b), the deep

158

layers (60-100cm) exhibited higher PCP dechlorination extents (average value of 93%)

159

than the upper layers (0-60cm) (average of 24%). When supplied with sufficient

160

carbon (Figure 1a), PCP dechlorination began after 7 days incubation for soils

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collected from the 40-100 cm depths and continued for approximately 84 days, after

162

which PCP was almost totally removed with an average residual amount of 1.75% in

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soil slurries. PCP was quantitatively transformed to 2,3,4,5-TeCP by initial

164

dechlorination at the ortho-position, and by a subsequent second dechlorination to

165

produce 3,4,5-TCP (Figure 1). Traces of 3,4-DCP and 3-MCP were also detected at

166

the end of incubation period (Figure 1c, data not shown).

167

Isotope Fractionation Observed in PCP Decreasing Steps

168

Carbon isotope enrichment effects were detected during PCP dechlorination to

169

examine the extent and the mechanism of PCP dechlorination under anaerobic

170

conditions (Figure 1d). The associated C isotope enrichment factor (εC) was

171

calculated at 42 and 84 days in treatments both with and without acetate in all five soil

172

profiles, based on eq 1. The value of εC was equal to -3.01±0.1‰, and showed a

173

highly linear correlation with changes in the C isotope composition and changes in the

174

concentration of residual fraction of PCP during the degradation process (R2 = 0.97, p

175

< 0.001). The high linear consistency confirms that isotope fractionation did not differ

176

among the treatments and soil layers, which contrasts with the results showing

177

variable reaction rates for PCP reductive dechlorination as described above.

178

Dynamic Processes of Environmental Variables and Their Correlations

179

Dynamic changes occurred in all the basic physicochemical variables that were

180

associated directly or indirectly with soil redox processes (Cl-, DOC, DON, FeHCl, Eh,

181

CO2, Fe(II), Mn(II), SO42-, RSCs, CH4) during the 84 day anaerobic incubation

182

(Section S-3, Figure S3-S13 and Table S2). As expected, the reduction of Fe(III),

183

SO42 and CO2 was strongly driven by addition of acetate as an electron donor and

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carbon substrate and was observed in all five soil layers during 84 days of anaerobic

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incubation. However, there was also a significant interaction in which the presence of

186

PCP as a soil contaminant had differential effects on the utilization of these three

187

terminal electron acceptors (Figure S9-S13).

188

The Multiple Correspondence Analysis (MCA) method was used to describe,

189

explore and summarize the information contained within the large data set of these

190

dynamic processes, based on three categorical indexes of soil profiles, experimental

191

treatments and environmental factors. The first two principal dimensions of (MCA)

192

explained 49.4% (MCA1) and 46.3% (MCA2) of the total inertia, respectively (Figure

193

2a), and the three indexes were clearly separated within the first and second

194

dimensions. Axis 1 separated the treatment with or without acetate and axis 2

195

established a separation between treatments with and without PCP. Different soil

196

profiles were clearly clustered near the origin area, which indicated high

197

correspondence among the samples according to soil layer/depth. Compared to the

198

soil layers, different treatments that separated at four different quadrants exhibited

199

large variances between each other. The variables of “cDOC”, “CH4” and “CO2” that

200

related to carbon source were clustered with the treatment of “Ace” and “PCP+Ace”.

201

Fe(III) reduction was predominant in deep soil layers treated with acetate addition,

202

and the SO42- reducing processes were strongly gathered with the PCP treatment in

203

the fourth quadrant.

204

Detailed relationships between PCP dissipation and environmental variables in

205

the soil profile were further examined by Pearson pairwise correlations (Figure S14).

206

Residual PCP was significantly and negatively correlated with “Fe2+”, “RSCs” and

207

“CH4”, three important final products of electron transport processes (p < 0.001).

208

Additionally, strong negative correlations between “PCP” and “FeHCl” or Fe(II),

209

implied that high rates of PCP dechlorination would occur under conditions with

210

abundant iron and an active Fe(III)-reducing microbial community. PCP dissipation

211

could also be accelerated in soils with a higher organic carbon content, as indicated by

212

the negative correlation between “PCP” and “cDOC”. Likewise, both “Eh” and “pH”

213

were inversely correlated with “PCP” (p SO42- > CO2.

402

Although the reaction time and the redox potential of PCP reductive dechlorination

403

were similar to SO42- reduction, Villemur et al.26 suggested that Desulfitobacterium

404

spp. has the advantage of using H2 below the threshold concentration that would allow

405

sulfate reduction and methanogenesis. Therefore, it was inferred that the

406

dechlorination process should slightly prevail over sulfate reduction under anaerobic

S-3).

Conversely,

processes involving

SO42- reduction and

PCP

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conditions, and thus the sequences including PCP would be in the order of NO3- >

408

Fe(III) > PCP ≥ SO42- > CO2.

409 410

Influences of Acetate / PCP Addition on the Main Electron Transporting

411

Processes

412

Biogeochemical cycling processes are generally considered to involve

413

sequential redox reactions of redox-active elements such as NO3-, Fe(III), SO42- and

414

CO23. Acetate, which is expected to be the most important product of fermentation,

415

can be widely used by multiple microbial populations35,43. In long-term flooded soil,

416

acetate serves as an important carbon source and electron donor for acetate-utilizing

417

bacteria, and is either converted to methane in methanogenic environments or to CO2

418

with NO3-, Fe(III), SO42- as alternative electron acceptors

419

acetate dramatically accelerated almost all of the reducing processes (Figure S8-S13).

420

The concentration of CO2 (Figure S8) that was generated by metabolism of acetate

421

was 20-fold greater than that for CH4 (Figure S13). This implies that ferric iron

422

reducers and sulfate reducers were active enough to outcompete methanogens for

423

acetate and diverted reducing equivalents away from CH4 production towards Fe(III),

424

SO42- and PCP reduction. MCA analysis of each treatment viewed by each index

425

showed that the distance between the treatment of “Ace+PCP” and “Ace” was closer

426

than that with the “None” and “PCP” treatments, and was highly clustered with the

427

variables of “cDOC” and “CO2” rather than “PCP” (Figure 2a). This suggested that

428

the influence of PCP in the treatment of “Ace+PCP” was relatively masked and the

429

corresponding biogeochemical reactions were more influenced by the addition of a

430

carbon source than by the presence of PCP.

44-47

. Here, the addition of

431

SO42- reduction was obviously suppressed by the PCP addition, as evidenced by

432

the sharp decrease in SO42- reduction rates following the addition of PCP (Figure S11).

433

Based on the high consistency of the decreased reduction rate of SO42- and the

434

depressed abundance of SO42- reducers when exposed to PCP, it was concluded that

435

the toxicity of PCP was the main reason for the inhibited reduction of SO42-.

436

Additionally, methanogenesis is usually the terminal process of organic matter

437

metabolism in anoxic environments48-49. It was interesting that the flux of the CH4 in

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the treatment with PCP supplement was nearly twofold greater than that without PCP

439

(Figure S13). Enumeration of the related genes (Figure 3) suggested that the

440

inhibition of sulfate reducers by the PCP toxicity resulted in a higher CH4 emission

441

rate, since a greater proportion of electrons diverted to methanogenesis, which

442

strongly supports the explanation for differences in CH4 emission between treatment

443

of “Ace+PCP” and “Ace”.

444 445

Enhanced Reductive Dechlorination of PCP Coupled with Fe(III) Reduction

446

Process

447

Analysis of the terminal electron-accepting processes, based on the redox state

448

changes of environmental variables and related microbial communities abundance,

449

indicated that Fe(III) reduction was predominant in this mangrove soil, as compared

450

to processes involving other electron acceptors. This was well evidenced by the

451

existence of abundant Fe(III) reducing bacteria and a significant correlation between

452

“Fe(II)” and PCP disappearance. Results between biotic and abiotic Fe(III) reduction

453

processes (Figure S2a) showed that the microbially mediated iron reducing process

454

was predominant in the soils under anaerobic conditions. Many studies on anaerobic

455

degradation of halogenated pollutants have shown that reductive dehalogenation

456

usually occurs under Fe(III)-reducing conditions50-52. Here, Pearson correlation

457

analysis revealed that “PCP” was significantly correlated to “FeHCl”, indicating that

458

the process of PCP reductive dechlorination was more favorable in soils that contain

459

abundant iron (Figure S14). Furthermore, the strong correlations of MCS with

460

“Fe(II)” and “PCP” showed that the bacterial communities were strongly structured

461

by Fe(III)-respiration and (de)halo-respiration processes. The observation that “PCP”

462

was highly correlated with the bacterial communities (Spearman’s coefficients, r = -

463

0.657, p < 0.01, Section S-6 and Table S5), and especially the population of Fe(III)

464

reducers “Geobacteraceae.sp” (Spearman’s coefficients, r = -0.843, p < 0.01)

465

strongly suggested that PCP dechlorination was promoted under conditions that also

466

support Fe(III) reduction.

467

A number of research groups have specifically investigated the synergistically

468

enhanced effect of reductive dehalogenation of halogenated OCs during Fe(III)-

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reduction, even though Fe(III) has been reported to be a strictly competitive electron

470

acceptor during anaerobic respiration52-54. Wei and Finneran53 showed that Fe(III)

471

reduction may actually help complete dechlorination of TCE to ethane, by controlling

472

the partial pressure of hydrogen, given that dissimilatory iron reducing bacteria can

473

reduce TCE to cis-dichloroethylene (cis-DCE). Actually, members of the genus of

474

Geobacter and Desulfuromonas within the Geobacteraceae family are metabolically

475

versatile with respect to their wide spectrum of electron donors and acceptors, and can

476

dehalogenate a wide range of halogenated aromatic and aliphatic compounds19,55-56.

477

The increase of Geobacteraceae in the qPCR and sequencing results in PCP spiked

478

soil slurries in our experiment (Figure 3, Figure S16) were likely due to growth of

479

Fe(III)-reducing bacteria that were coupled with PCP dechlorination. In this manner,

480

active Fe(III) reduction in Fe(III) rich soils should promote dechlorination of PCP to

481

lesser chlorinated chlorophenols.

482 483

IMPLICATIONS

484

Our results show that natural attenuation of PCP can occur in soils with no prior

485

history of anthropogenic contamination by organochlorines and that reductive

486

dechlorination of PCP at the ortho-position is the primary natural attenuation

487

mechanism in both surface and lower profile soil layers under long-term anaerobic

488

conditions. Fe(III) reduction was strongly associated with PCP dechlorination and

489

may be mainly mediated by dissimilatory iron reducing bacteria. The presence of PCP

490

significantly inhibited reduction of sulfate, as simultaneously evidenced by the low

491

reduction ratio and decreased numbers of sulfate reducers. This inhibition of SO42- by

492

PCP resulted in a corresponding release of methane as the community shifted to

493

methanogens. Additionally, providing sufficient carbon sources as electron donors

494

would effectively accelerate the dechlorination process through increasing the

495

diversity and abundance of dechlorinators or multifunctional microorganisms.

496

Though a systematic stoichiometric demonstration, our results provide a step

497

forward for improved understanding of natural biogeochemical processes for

498

remediation of soils by halogenated OCs such as PCP. Overall, the combination of

499

molecular and numerical biogeochemical approaches, as well as the CSIA method

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500

was helpful for understanding the reaction mechanisms between PCP and

501

environmental variables, and implied changes of microbial community structures

502

underlying these anoxic respiratory processes. Furthermore, the finding that acetate

503

played an important role in all of the studied redox processes including PCP

504

dechlorination, should guide future studies on the role of other electron donors

505

besides acetate for driving dechlorination of chlorinated OCs.

506 507

ASSOCIATED CONTENT

508

Supporting Information

509

Chemical and microbial community analysis methods, and details on the statistical

510

methods used to correlate dynamic processes with the environmental variables are

511

provided as online supporting information; Tables S1-S5 report the primer sets for

512

real time PCR, results for the kinetic analysis of accumulated Fe(II), and the statistics

513

generated by correlation analysis; Figures S1-S16 show the dynamic changes in

514

environmental processes during the 84 day incubation. This information is available

515

free of charge via the Internet at http://pubs.acs.org.

516 517

AUTHOR INFORMATION

518

Corresponding Author

519

∗

520

[email protected]; Corresponding author address: Institute of Soil and Water Resources

521

and Environmental Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058,

522

China.

Phone: +86-571-8898-2069; Fax: +86-571-8898-2069; E-mail: [email protected];

523 524

Notes

525

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

527

This research was ﬁnancially supported by the National Natural Science Foundation

528

of China (41090284, 41322006), the National High Technology Research and

529

Development Program of China (863 Program, No. 2012AA06A203), and the

530

Fundamental Research Funds for the Central Universities.

531

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