Stable Carbon Isotope Fractionation During 1,2-Dichloropropane-to

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Stable carbon isotope fractionation during 1,2-dichloropropaneto-propene transformation by an enrichment culture containing Dehalogenimonas strains and a dcpA gene Lucía Martín-González, Siti Hatijah Mortan, Mònica Rosell, Eloi Parladé, Maira Martínez-Alonso, Nuria Gaju, Gloria Caminal, Lorenz Adrian, and Ernest Marco-Urrea Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00929 • Publication Date (Web): 25 Jun 2015 Downloaded from http://pubs.acs.org on June 29, 2015

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Environmental Science & Technology

TOC/Abstract art

1 2

Dehalogenimonas growth (qPCR)

Cl Cl C C C

3

1,2-dichloropropane

Detection of 1,2-DCPreductase gene dcpA

ε=-15.0 ± 0.7‰

C C C Propene

4 5 6 7 8 9 10

1 ACS Paragon Plus Environment


11

Stable carbon isotope fractionation during 1,2-dichloropropane-

12

to-propene transformation by an enrichment culture containing

13

Dehalogenimonas strains and a dcpA gene

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14 15 16

L. Martín-González1, S. Hatijah Mortan1, M. Rosell2, E. Parladé3, M. Martínez-Alonso3,

17

N. Gaju3, G. Caminal4, L. Adrian5, E. Marco-Urrea*,1

18 19

1

20

Carrer de les Sitges s/n, 08193 Bellaterra, Spain.

Departament d'Enginyeria Química, Universitat Autònoma de Barcelona (UAB),

21 22

2

23

Geologia, Universitat de Barcelona (UB), Martí Franquès s/n, 08028. Barcelona, Spain.

Departament de Cristal·lografia, Mineralogia i Dipòsits Minerals, Facultat de

24 25

3

26

Bellaterra, Spain.

Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193

27 28

4

29

Barcelona, Spain.

Institut de Química Avançada de Catalunya (IQAC) CSIC, Jordi Girona 18-26, 08034

30 31

5

32

Germany.

Helmholtz Centre for Environmental Research - UFZ, Permoserstraße 15, Leipzig,

33 34

*

Phone: +34 5812694; fax: +34 5812013; e-mail: [email protected].


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36

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ABSTRACT

37 38

A

stable

enrichment culture

39

stoichiometrically dechlorinated 1,2-dichloropropane (1,2-DCP) to propene. Sequential

40

transfers in defined anaerobic medium with the inhibitor bromoethanesulfonate

41

produced a sediment-free culture dechlorinating 1,2-DCP in the absence of

42

methanogenesis. Application of previously published genus-specific primers targeting

43

16S rRNA gene sequences revealed the presence of a Dehalogenimonas strain, and no

44

amplification was obtained with Dehalococcoides-specific primers. The partial

45

sequence of the 16S rRNA amplicon was 100% identical with Dehalogenimonas

46

alkenigignens strain IP3-3. Also, dcpA, a gene described to encode a corrinoid-

47

containing 1,2-DCP reductive dehalogenase was detected. Resistance of the

48

dehalogenating activity to vancomycin, exclusive conversion of vicinally chlorinated

49

alkanes, and tolerance to short-term oxygen exposure is consistent with the hypothesis

50

that a Dehalogenimonas strain is responsible for 1,2-DCP conversion in the culture.

51

Quantitative

52

Dehalogenimonas 16S rRNA genes copies in the culture and consumption of 1,2-DCP.

53

Compound specific isotope analysis revealed that the Dehalogenimonas-catalyzed

54

carbon isotopic fractionation (ǫCbulk) of the 1,2-DCP-to-propene reaction was -15.0 ±

55

0.7‰ under both methanogenic and non-methanogenic conditions. This study

56

demonstrates that carbon isotope fractionation is a valuable approach for monitoring in

57

situ 1,2-DCP reductive dechlorination by Dehalogenimonas strains.

PCR

showed

a

derived

positive

from

Besòs river estuary sediments

correlation

between

the

number

of

58


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59

1. INTRODUCTION

60 61

1,2-dichloropropane (1,2-DCP) has been used predominantly as a chemical intermediate

62

in the production of tetrachloromethane (carbon tetrachloride) and perchloroethene, lead

63

scavenger for antiknock fluids and solvent. According to the National Primary Drinking

64

Water Regulations established by the U.S. Environmental Protection Agency (EPA), 1

65

1,2-DCP can increase risk of cancer so a maximum contaminant level in drinking water

66

of 5 µg L-1 is legally set for public water systems. Today, 1,2-DCP is a risk for the

67

environment and drinking water quality especially at historically contaminated sites.

68

Under aerobic conditions, 1,2-DCP can be partially co-metabolized to less-

69

chlorinated alkanes. For instance, Pseudomonas sp. strain DCA1 oxidized co-

70

metabolically 1,2-DCP to 2,3-dichloro-1-propanol and 2-chloroethanol during growth

71

on 1,2-dichloroethane2. However, 1,2-DCP exerted a strong inhibitory effect on the

72

growth of this Pseudomonas strain, probably due to a transient toxic intermediate.2

73

Similarly, resting cells of the methanotroph Methylosinus trichosporium OB3b

74

expressing soluble methane monooxygenase catalyzed the transformation of 1,2-DCP to

75

2,3-dichloro-1-propanol, 1-chloro-2-propanol and 2-chloro-1-propanol.3 So far,

76

complete dechlorination of 1,2-DCP to propene or propane has only been demonstrated

77

for organohalide respiring bacteria (OHRB), which use halogenated compounds as

78

terminal electron acceptors during electron transport-based energy conservation.4 To

79

date, few isolated OHRB have been described to derive energy for growth from

80

dechlorination of 1,2-DCP, including Dehalogenimonas alkenigignens strain IP3-3,5

81

Dehalogenimonas lykanthroporepellens strain BL-DC-8 and strain BL-DC-9,6

82

Dehalococcoides mccartyi strain RC and strain KS,7 and Desulfitobacterium

83

dichloroeliminans strain DCA1.8 A Dehalobacter strain was involved in the reductive



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dechlorination of 1,2-DCP in a consortium.9 Although these bacteria share several

85

common phenotypic features including growth under anaerobic conditions, the use of

86

halogenated compounds as respiratory electron acceptors and hydrogen as electron

87

donor, isolated Dehalogenimonas strains differ from other 1,2-DCP-transforming

88

isolates in their exclusive utilization of polychlorinated alkanes as halogenated electron

89

acceptors. Recently, a gene designated dcpA was identified as encoding the reductive

90

dehalogenase

91

Dehalococcoides.10

that

catalyzes

the

dechlorination

of

1,2-DCP-to-propene

in

92

Advances in the fundamental understanding of reductive dechlorination

93

reactions can contribute to an improved assessment of these degradation processes in

94

contaminated subsurface environments. In the last 15 years, compound specific stable

95

isotope analysis has shown the potential of using stable isotope fractionation to confirm

96

and quantify in situ bioremediation and elucidate transformation pathways of many

97

pollutants including chlorinated organic contaminants.11,12 Dichloroelimination is

98

reported to be the major transformation process for 1,2-DCP by OHRB.

99

reaction involves the simultaneous removal of two chlorines from adjacent carbon

100

atoms with the formation of a carbon-carbon double bound, leading to the production of

101

propene from 1,2-DCP. The transformation of 1,2-DCP to propene can proceed via

102

either a stepwise mode (involving a transition state with one C-Cl bond) or a concerted

103

mode (involving the simultaneous cleavage of both C-Cl bonds). In an attempt to

104

elucidate which biochemical mechanism was involved, Fletcher et al.,15 calculated the

105

apparent kinetic isotope effect (AKIE) values simulating both reaction scenarios

106

(stepwise versus concerted mode) from the first carbon isotopic fractionation values (ε)

107

obtained from two different enrichment cultures containing Dehalococcoides mccartyi

108

strains RC and KS. Then the values were compared to the theoretical maximum carbon

8,13,14

This


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109

primary AKIE (“semiclassical Streitwieser Limits”) for the cleavage of a C-Cl bond.

110

However, the estimated AKIEs assuming stepwise or concerted reaction were not

111

conclusive to demonstrate the reaction mechanism of 1,2-DCP dichloroelimination. The

112

ε values obtained for the two Dehalococcoides mccartyi strains in their respective 1,2-

113

DCP-to-propene consortia were statistically identical (-10.8 ± 0.9‰ and -11.3 ± 0.8‰,

114

respectively).

115

The aim of this study was to get more insight into the microbial processes

116

involved in chloroalkane dechlorination and strengthen the prospect of applying

117

compound specific stable isotope analysis as a tool to demonstrate in situ

118

biodegradation. We established an enrichment culture derived from sediments collected

119

in the Besòs River estuary (Barcelona) that dechlorinates vicinally chlorinated alkanes.

120

This location was chosen because sediments of this coastal area have been historically

121

contaminated with short-chain chlorinated paraffins,16 providing a potential niche for

122

OHRB. Our results demonstrate that Dehalogenimonas spp. were responsible for 1,2-

123

DCP dechlorination and carbon isotopic fractionation differed from that described for

124

Dehalococcoides mccartyi strains although the dcpA gene involved in the 1,2-DCP-to-

125

propene transformation in Dehalococcoides strains was also present in our culture.

126 127

2. MATERIALS AND METHODS

128 129

2.1. Sampling and cultivation

130

Inocula were taken from sediments of the Besòs river estuary (Spain). The samples were

131

collected from layers 15 cm below the surface. Sediments were transported to the lab,

132

transferred to an anaerobic glovebox and used to set up microcosms on the same day.

133

Each microcosm consisted of 6 g of sediment (wet weight) in 100 mL glass serum



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134

bottles containing 65 mL of a sterilized anaerobic synthetic medium previously used to

135

grow Dehalococcoides mccartyi strain CBDB1.17 It contained vitamins, trace elements,

136

5 mM sodium acetate as carbon source, and as reducing agent either 0.8 mM titanium

137

(III) citrate (0.8 mM titanium (III), 1.6 mM citrate) or Na2S × 9 H2O and L-cysteine (0.2

138

mM each), as indicated. The serum bottles were sealed with Teflon-coated butyl rubber

139

septa and aluminum crimp caps and gassed with N2/CO2 (4:1, v/v, 0.2 bar overpressure)

140

and H2 (added to an overpressure of 0.4 bar). 1,2-DCP was added with a syringe from a

141

3.2 mM stock solution in acetone to a nominal concentration of 50 µM. Microcosms

142

were prepared at least in triplicate and incubated at 25 ºC in the dark in static

143

conditions. Microcosms that depleted the initial dose of 1,2-DCP were reamended with

144

the same amount of the electron acceptor and transferred to fresh medium (10% v/v)

145

when approximately 80% of the initial dose of 1,2-DCP was consumed.

146

For isotopic analysis, several parallel incubations from the same inoculum were

147

prepared at the same time and sacrificed at different time points. Three types of controls

148

were included at least in duplicate: i) controls containing heat-killed inoculum and 1,2-

149

DCP, ii) live controls without 1,2-DCP, and iii) abiotic controls containing the growth

150

medium with 1,2-DCP without inoculum, to control losses, abiotic transformations, and

151

the transfer of compounds from previous degradation experiment with the inoculum or

152

potential impurities from the stock solution, respectively.

153 154

2.2. Analytical methods

155

Chlorinated compounds, propene and methane were detected by analyzing 0.5 mL

156

headspace samples respectively taken with a 1.0 mL pressure-lock precision analytical

157

syringe (Vici, USA) from the serum bottles. All compounds were identified using

158

retention times of chemical standards. A gas chromatograph (GC) model 6890N


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(Agilent Technologies; Santa Clara, CA, USA) equipped with a DB-624 column (30 m

160

× 0.32 mm with 0.25 µm film thickness; Agilent Technologies) and a flame ionization

161

detector (FID) was used to analyze all volatile organic compounds. Helium was used as

162

the carrier gas (0.9 mL min-1). The injector and detector temperatures were set at 250 °C

163

and 300 °C respectively. After the injection of the sample (split ratio=2), the initial oven

164

temperature (35 °C) was held for 3 min and then ramped at 10 °C min-1 to 240 °C,

165

which was held for 4 min. Peak areas were calculated using Millennium/Empower

166

software (Waters, Milford, MA, USA). Calibration was based on aqueous standards,

167

with the same liquid and headspace volumes as in the microcosms. Results are

168

presented as nominal concentrations (µmol L-1 of liquid volume). Propene was analyzed

169

using an identical GC-FID equipped with a HP Plot Q column (30 m × 0.53 mm with 40

170

µm film thickness, Agilent Technologies). The oven temperature was fixed at 150 ºC,

171

the injector temperature at 250 ºC and the detector temperature at 260 ºC. Run time

172

lasted 6 minutes.

173

Methane concentration was analyzed using a GC HP 5890 (Agilent

174

Technologies, Palo Alto, USA) with a thermal conductivity detector (TCD) equipped

175

with a Porapack Q column (3 m x 3.2 mm, Sigma-Aldrich, Barcelona, Spain) using

176

helium at 338 kPa as the carrier gas. The oven temperature was fixed at 70 ºC, the

177

injector temperature at 150 ºC and the detector temperature at 180 ºC. Run time was 3

178

minutes.

179

When needed, identification of transformation products was done using a gas

180

chromatograph equipped with a mass selective detector (Agilent 5975C + 7890 Series

181

GC-MS). Compounds were separated using a DB-624 column (60 m × 0.25 mm, film

182

thickness 1.4 µm, Agilent Technologies). Carrier gas was helium at a flow rate of 0.8

183

mL min-1. The temperature was hold for 2 min at 50 °C, raised to 170 °C at a rate of 3 9 ACS Paragon Plus Environment


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°C min-1 and finally to 230 ºC at a rate of 8 °C min-1. The transfer line to the mass

185

spectrometer was maintained at 235 °C. Mass spectra were obtained after ionization by

186

electronic impact at 70 eV and at a multiplier voltage of 1379 V. Data was collected at

187

m/z values between 33 and 300. Mass spectra obtained were compared with the NIST

188

98 MS Library Database and with the generated mass spectra of standards.

189

Diclofenac and triclosan concentrations were measured using high performance

190

liquid chromatography (HPLC). Liquid samples (1 mL) from experimental bottles were

191

diluted 1:1 (v:v) with acetone as a solvent before mixing on a vortex (Zx3,Velp

192

Scientifica, Italy). Next, the samples were filtered (Millex-GV, PVDF, 0.22 µm,

193

Millipore) and analysed using a Dionex 3000 Ultimate HPLC (Barcelona, Spain) that

194

was equipped with a UV detector at 277 nm. The column temperature was 30 ºC, and a

195

sample volume of 10 µL was injected from a Dionex autosampler. Chromatographic

196

separation was achieved using a GraceSmart RP-18 column (250 x 4.6 mm, particle size

197

of 5 µm). The mobile phase consisted of a 0.1% formic acid solution (A) and

198

acetonitrile (B). The analysis was performed isocratically (30% A) at 1 mL min-1. The

199

retention times for diclofenac and triclosan were 4.3 min and 5.4 min, respectively. The

200

quantification limit for both compounds was 2 mg L-1.

201

The presence of bromide ions was evaluated on a Dionex ICS-2000 ion

202

chromatography system equipped with an IonPac AS18 anion-exchange column. The

203

column was operated at a temperature of 30 °C and a flow rate of 1 mL min-1. The

204

injection volume was 25 µL. The potassium hydroxide concentration of the eluent

205

varied from 25 mM to 50 mM along the 10 min analysis.

206

For isotopic analysis, 6 mL of NaOH was added to parallel incubations to stop

207

biological transformations at different time points and bottles were preserved at 4ºC

208

until analysis. Carbon isotope analyses of propene and 1,2-DCP were performed using a


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209

Agilent 6890 gas chromatograph (Palo Alto, CA, USA) equipped with a split/splitless

210

injector, coupled to a Delta Plus isotope ratio mass spectrometer through a GC-

211

Combustion III interface (ThermoFinnigan, Bremen, Germany). A headspace gas

212

sample with a volume between 0.6 and 1 mL, depending on propene concentration

213

measured previously, was injected by a gas syringe. For propene, the GC was equipped

214

with a HP-PLOT/Q column (30 m x 0.32 mm, 20 µm film thickness, Agilent

215

Technologies, Palo Alto, CA, USA). The injector was set at 220 ºC in split mode (1:10).

216

The oven temperature program was kept at 40 °C for 10 min, heated to 220 °C at a rate

217

of 15 °C min-1 and finally held at 220 °C for 10 min. Helium was used as a carrier gas

218

with a gas flow rate of 1.2 mL min-1. The retention time of propene was identified by

219

injecting 300 µL of a standard gas mixture (Supelco Scotty Analyzed Gases, C2-C6

220

Olefins, Sigma-Aldrich, St. Louis, Missouri, USA) containing ethene, propene, 1-

221

butene, 1-pentene, 1-hexene each at 100 mg L-1. Once the δ13C of propene was

222

analyzed, liquid aliquots were removed from the experimental bottles and placed in 20-

223

mL vials filled with 10-mL aqueous phase (samples were diluted or not in Milli-Q water

224

depending on the 1,2-DCP concentration) and containing a 30 mm PTFE-coated stir

225

bar. This solution was stirred at room temperature and 1,2-DCP was extracted during 20

226

minutes by headspace solid-phase micro-extraction (HS-SPME) using a manual sampler

227

holder equipped with a 75-µm Carboxen-PDMS fiber (Supelco, Bellefonte, PA, USA).

228

For 1,2-DCP, the GC was equipped with a Supelco SPB-624 column (60 m × 0.32 mm,

229

1.8 µm film thickness; Bellefonte, PA, USA). The injector was set at 220 ºC in split

230

mode (1:10). The oven temperature program was kept at 60 °C for 2 min, heated to 220

231

°C at a rate of 8 °C min-1 and finally held at 220 °C for 5 min. Helium was used as a

232

carrier gas with a gas flow rate of 1.8 mL min-1. Several 1,2-DCP aqueous control

233

standards were prepared daily from the same pure 1,2-DCP standard which was also



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234

used for the cultures (standard stock solutions were prepared first in HPLC grade

235

methanol) and analyzed on the same days as the samples to ensure accuracy of the

236

isotopic measurements. All the controls injected in different replicas, days and

237

concentrations (from 30 to 300 µg L-1) had an average 1,2-DCP-δ13C values of -29.3 ±

238

0.5‰ (n = 34).

239

Carbon isotope ratios are reported in delta notation (δ13C) relative to an international

240

standard (VPDB, Vienna Pee Dee Belemnite) and expressed in parts per mil (‰)

241

13 ‰ =

− 1 × 1000

are the isotope ratios (13C/12C) of the sample and the standard,

242

Where

243

respectively. A simplified Rayleigh equation for a closed system was used to quantify

244

the isotopic fractionation:11

245

and

(1)

ɛ ln = ln 0 0 1000

(2)

246

where the isotopic fractionation (ε) describes the relationship between changes in

247

carbon isotopic composition

248

concentrations (C) along the time course (t) with respect to the initial concentration (0).

249

Most of the measurements were run in duplicate, and the one standard deviation (1σ) of

250

the δ13C values obtained was below or equal to ±0.5‰.18

251

Position-specific carbon isotopic fractionation (ǫreactive) values, which are corrected for

252

the presence of nonreactive positions, were calculated for both stepwise and concerted

253

reactions according to Elsner et al.11 as done by Fletcher et al.15, where n is the number

254

of carbon atoms in the molecule (in the case of 1,2-DCP, n =3) and x is the number of

255

carbon atoms in the reactive position (in the case of a stepwise reaction, x = 1 and in the

256

case of a concerted reaction, x = 2) by plotting again the modified Rayleigh equation:

⁄0 = 13 + 1000 ⁄ 13 0 + 1000

and the

257 12 ACS Paragon Plus Environment

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258

1000 + 13 0 + "⁄# ∆ 13 ɛ& '() ln ! %= ln 13 0 1000 + 0 1000

(3)

259

Then, the apparent kinetic carbon isotope effect (AKIE) for each case was calculated

260

according to:

261

*+,- = 1⁄.1 + / × ɛ& '() ⁄1000 0

(4)

262

where z, the number of indistinguishable reactive sites, is a correction for the effects of

263

intramolecular competition (in the case of 1,2-DCP, z = 1).

264

Carbon isotopic mass balance and the associated concentration-weighted average

265

δ13Csum is calculated similar to the sequential reductive dechlorination of chlorinated

266

ethenes19,20 but only considering 1,2-DCP as parental compound and propene as final

267

product:

268

13 1 ‰ = #1,2−45 13 1,2−45 + #&6" 13 &6"

(5)

269

where x is the molar fraction of each compound relative to the total molar mass (1,2-

270

DCP plus propene) at each time. If propene is not further transformed, the δ13Csum

271

remains constant over the 1,2-DCP dichloroelimination. As a certain amount of propene

272

was transferred with the inoculum from the previous experiment (initial propene

273

detected and quantified in live controls without 1,2-DCP), the

274

corresponding to newly generated propene in equation 5 was calculated as follows:

275

13 &6" =

13 1&

&6"

− #("((

#7"&

&6"

&6"

13 ("((

&6"

(6)

276 277

2.3. DNA extraction and PCR

278

Genomic DNA was extracted from 50 mL of the consortium using an UltraClean water

279

DNA isolation kit (MoBio, Carlsbad, CA). Amplification of bacterial 16S rRNA genes

280

was conducted with two sets of primers. The first primer combination (BL-DC-142f and



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281

BL-DC-1351r) was specific for the genus Dehalogenimonas.21 The second primer set

282

specifically targeted the genus Dehalococcoides (Dch1F and Dch264R).22 Genomic

283

DNA was also analyzed for the presence of the dcpA gene. Primers dcpA-360F and

284

dcpA-1449R10 designed for conventional PCR were used. Genomic DNA from

285

Dehalogenimonas lykanthroporepellens strain BL-DC-9 (=ATCC BAA-1523 = JCM

286

15061) and Dehalococcoides mccartyi strain CBDB1 were used as positive controls.

287

Each 50 µL reaction mixture contained 50 ng of template DNA, 1x PCR buffer (20 mM

288

Tris/HCl, pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 200 µM of each deoxynucleoside

289

triphosphate, 0.5 µM of each primer and 2.5 U of Taq DNA polymerase (Invitrogen,

290

Carlsberg, CA, USA). The thermal programs used for PCR amplification of

291

Dehalogenimonas and dcpA gene were previously described.10,21 For primer set Dch1F

292

and Dch264R, the program used an initial denaturation at 95 °C for 5 min and then 35

293

cycles at 95°C for 30 s, the desired annealing temperature (59 °C) for 30 s, and

294

extension at 72°C for 60 s, followed by a final extension step at 72 °C for 7 min.

295

Amplicons were analyzed by electrophoresis in a 2 % (wt/v) agarose gel at 75 V for 40

296

min. Primers used in the second amplification in the nested PCR approach were 341f-

297

GC and 907r. Temperature cycling was done as described previously.23 Sequences of

298

the different oligonucleotide primer sets used in this study are given in Table S2.

299 300

2.4 Denaturing Gradient Gel Electrophoresis (DGGE) analysis and sequencing

301

Five-hundred ng of PCR product from nested PCR using the primer sets BL-DC-

302

142f/BL-DC-1351r and 341f-GC/907r were loaded onto a denaturing gradient gel.

303

DGGE was carried out using a Bio-Rad DCode system, as described,23 in a 6%

304

polyacrylamide gel with 30-70% denaturant gradient (100% denaturant contained 7 M

305

urea and 40% v/v deionized formamide). Electrophoresis was performed at 60 ºC with a

306

constant voltage of 75 V for 16 h. The gels were stained with ethidium bromide (0.5 µg 14 ACS Paragon Plus Environment

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mL-1), then inspected under UV illumination and photographed. Prominent bands were

308

excised from the gels, reamplified, and then purified using the PCR Clean up Kit

309

(MoBio Laboratories) for subsequent sequencing.

310

Sequencing reactions were performed by Macrogen (South Korea) using the Big Dye

311

Terminator v3.1 sequencing kit; reactions were run in an automatic capillary type ABI

312

3730XL analyzer-96. Sequences were first screened to detect potential chimeric

313

artifacts

314

(http://www.mothur.org/wiki/Download_mothur)24 and then compared to those

315

deposited in the GenBank nucleotide database using the BLASTN program.25 The 16S

316

rRNA gene sequences determined in this study are available at the GenBank database

317

under accession numbers KP780280 through KP780282. Each band designation

318

includes a code specifying its origin (BREd, Besòs River Estuary Dehalogenimonas)

319

followed by a number indicating the order in which the sequence was isolated from the

320

gel.

using

the

Chimera.uchime

program

in

Mothur

1.33.3

321 322

2.5 Quantitative PCR (qPCR)

323

The qPCR assays were performed with DNA extracted from cultures growing in

324

parallel consuming different amounts of 1,2-DCP. Primer set mod-BL-DC-1243f and

325

BL-DC-1351r21 was used in qPCR to quantify Dehalogenimonas 16S rRNA gene

326

copies in the consortia. qPCR was performed using a CFX96 Real-Time System (Bio-

327

Rad) in 20 µL total reaction volumes containing 1x ssoAdvanced Universal SYBR®

328

Green Supermix (Bio-Rad), 0.5 µM of each primer and 36 ng of sample DNA. The

329

amplification program used was reported previously.21 A melting curve analysis to

330

assess product specificity followed each PCR reaction. Melting curves were generated

331

from 65 to 95 ºC with increments of 0.5 ºC each cycle and a dwell time at each



Page 16 of 35

332

temperature of 5 s. Samples and non-template controls were analyzed in triplicates and

333

the latter were included in each assay. Each calibration curve was prepared using

334

purified PCR product of a partial 16S rRNA gene (1199 bp) from Dehalogenimonas

335

lykanthroporepellens BL-DC-9T. Six serial dilutions were prepared independently in

336

triplicates and concentrations were determined with a NanoDrop spectrophotometer

337

(Thermo Fisher Scientific). Gene copies per qPCR reaction and PCR amplification

338

efficiency were calculated as described previously.26 PCR amplification efficiency

339

ranged from 93.2% to 102.3%.

340 341

3. RESULTS AND DISCUSSION

342

3.1. Enrichment of dehalogenating bacteria

343

Sediments collected from the Besòs river estuary were incubated in reduced medium

344

containing 50 µM 1,2-DCP. In the first transfer, hydrogenolysis of 1,2-DCP to 1-

345

chloropropane and minor amounts of 2-chloropropane was the predominant reaction in

346

most of the cultures. Hydrogenolysis of chloroalkanes was an unexpected reaction

347

which has been reported only once before,13 and therefore it was specifically followed.

348

A decrease in the production of chloropropanes in the subsequent transfers was

349

observed, and titanium (III) citrate was replaced by Na2S and L-cysteine (0.2 mM each)

350

in parallel cultures to test whether titanium (III) citrate exerted inhibition to the

351

dechlorinating population catalyzing hydrogenolysis. Production of chloropropanes was

352

favored in the cysteine-sulfide medium but after the fourth transfer hydrogenolytic

353

activity was lost and propene became the unique identified transformation product.

354

Although we corroborated that hydrogenolysis of chloropropanes is possible, the

355

identity and characteristics of the bacteria catalyzing this process remained unknown.

356

Similarly to the results showed by Löffler et al.,13 hydrogenolysis was not observed in 16 ACS Paragon Plus Environment

Page 17 of 35


357

sediment-free cultures, which is consistent with the hypothesis that microorganisms

358

catalyzing this reaction may be favored in sedimentary environments.

359

Dihaloelimination of 1,2-DCP to propene was sustained in subsequent transfers

360

inoculating 10% (v/v) from the original microcosms using the L-cysteine-sulfide

361

reducing agent. A sediment-free culture was obtained after five consecutive transfers of

362

the supernatant to fresh medium. The repeated addition of 1,2-DCP led to faster

363

dechlorination rates suggesting that dichloroelimination was supporting growth of the

364

dechlorinating bacteria (Fig. S1A). The stoichiometric relationship between 1,2-DCP

365

consumed and propene produced revealed a closed molar balance and excluded the

366

production of alternative metabolites (Table S1). Neither dechlorination of 1,2-DCP nor

367

propene production were detected in the abiotic and heat-killed controls indicating that

368

the reaction was biotically mediated.

369

As shown in Fig. S1A, methane was produced parallel with the dechlorination of

370

1,2-DCP. To investigate the role of methanogens in dichloroelimination of 1,2-DCP, we

371

tested the effect of bromoethanesulfonate (BES) at two different concentrations (5 and

372

25 mM) and of 1,2-dibromopropane (10 µM) on dechlorination activity. The latter was

373

initially tested as a possible electron acceptor (see below) but instead of that, we

374

observed that it completely inhibited methane production in our cultures. Methane

375

production was not inhibited at 5 mM BES , but it completely ceased at a concentration

376

of 25 mM with no negative effect on dechlorinating activity (Fig. S1B). Addition of 10

377

µM of dibromopropane completely inhibited both methanogenic activity and 1,2-DCP

378

dechlorination. The addition of vancomycin (5 mg L-1), an inhibitor of peptidoglycan

379

cell-wall biosynthesis, provoked an increase in the dichloroelimination rate of 1,2-DCP.

380

The consortia containing vancomycin were transferred for more than eleven subsequent

381

transfers (10% v/v) without losing this dechlorinating activity.



Page 18 of 35

382 383

3.2. Dehalogenation of alternative electron acceptors

384

Besides 1,2-DCP, we tested the potential of the consortium to reductively dechlorinate

385

various

386

dichloroethane,

387

(tetrachloroethene,

388

(monochlorobenzene, 1,2,4-trichlorobenzene, diclofenac, and triclosan), bromoalkanes

389

(1,6-dibromohexane, 1,2-dibromopropane) and chloroform by adding them at a

390

concentration between 5 and 50 µM. After one month of incubation, the concentrations

391

of the halogenated compounds were analyzed by GC. Debromination activity was tested

392

by measuring bromide concentration by ion chromatography. Dehalogenation was only

393

observed in cultures containing vicinally chlorinated alkanes. Thus, 1,2,3-

394

trichloropropane was converted to allyl chloride and allyl alcohol; 1,1,2-trichloroethane

395

was converted to vinyl chloride; and 1,2-dichloroethane to ethene.

chlorinated

alkanes

(1,2,3-trichloropropane;

1-chloropropane

and

trichloroethene,

1,1,2-trichloroethane;

2-chloropropane),

chlorinated

trans-dichloroethene),

1,2-

alkenes

chloroaromatics

396 397

3.3. Identification of Dehalogenimonas spp and the dcpA gene

398

To

399

Desulfitobacterium and Dehalobacter) have been implicated in the dechlorination of

400

1,2-DCP to propene. The presence of OHRB belonging to the genus Dehalobacter and

401

Desulfitobacterium in our cultures was ruled out because they cannot grow in the

402

presence of vancomycin. In addition, morphological evidence supported the

403

involvement of either Dehalogenimonas or Dehalococcoides since microscopic

404

observations of cultures fed with several 1,2-DCP additions showed predominantly

405

irregular cocci, but not rod-shaped cells as those described for Desulfitobacterium and

406

Dehalobacter spp (data not shown). In order to determine if known OHRB were present

date,

four

OHRB

populations

(Dehalogenimonas,

Dehalococcoides,


Page 19 of 35


407

in the dehalogenating consortium we performed PCR reactions with genus-specific

408

primers for Dehalococcoides and Dehalogenimonas 16S rRNA. The tested PCR primers

409

targeting for Dehalococcoides spp failed to produce an amplicon, but confirmed the

410

presence of Dehalogenimonas spp in our culture (Fig. S2A and S2B). Furthermore,

411

nested PCR followed by DGGE allowed the visualization of a prominent band (Fig. S3).

412

Several band replicates were excised from the gel, sequenced, and partial 16S rRNA

413

gene sequences obtained (accession numbers from KP780280 to KP780282) showed

414

100% identity with Dehalogenimonas alkenigignens strain IP3-3.

415

The

hypothesis

that

Dehalogenimonas

strains

are

involved

in

the

416

dichloroelimination of 1,2-DCP in these enrichment cultures was consistent with three

417

complementary lines of evidence. First, we tested the oxygen tolerance of the OHRB

418

contained in our culture by exposing the inoculum to air until the redox indicator

419

resazurin turned pink, and afterwards it was injected into fresh reduced medium. These

420

cultures grew at the same dechlorination rate than positive controls not exposed to

421

oxygen, which is consistent with the oxygen tolerance observed for Dehalogenimonas14

422

but disfavors the involvement of Dehalococcoides species due to their strong sensitivity

423

to short-term oxygen exposure.22 We repeated the short-time exposure of the inoculum

424

to oxygen for three serial transfers with identical results. Secondly, the OHRB present

425

in our culture solely dechlorinate vicinally chlorinated alkanes. This strong substrate

426

specialization is one of the remarkable characteristics observed for Dehalogenimonas

427

strains.5,6 Although it has been described by others that growth of a Dehalogenimonas

428

population in a consortium was coupled to reductive dechlorination of trans-

429

dichloroethene to vinyl chloride,22 our culture could not transform chlorinated ethenes

430

within one month of incubation. Thirdly, we monitored the abundance of

431

Dehalogenimonas 16S rRNA gene copies using quantitative PCR (qPCR) to determine



Page 20 of 35

432

whether 1,2-DCP dechlorination was coupled to Dehalogenimonas growth in the Besòs

433

river cultures. The addition of several successive doses of 1,2-DCP when all the

434

electron acceptor was consumed (Fig. 1A) resulted in a concomitant increase in the

435

number of Dehalogenimonas 16S rRNA gene copies detected in the cultures (Fig. 1B).

436

No growth was observed in control cultures that received no 1,2-DCP, showing that 1,2-

437

DCP dechlorination was a growth-linked respiratory process. These findings were

438

consistent with the detection of the dcpA gene encoding 1,2-DCP reductive

439

dehalogenase in Dehalogenimonas strain BL-DC-9, Dehalococcoides mccartyi strain

440

RC and strain KS.10 As shown in Fig. S2C, a conventional PCR using dcpA-360F and

441

dcpA-1449R primers confirms the presence of a unique amplicon of the expected size

442

(1089 bp) when applied to genomic DNA from the consortium.

443 444

3.4. Effect of methanogenic activity on 1,2-DCP fractionation

445

Methanogenic archaea are often present in dehalogenating communities and compete

446

with OHRB for hydrogen and acetate, but some populations can also synthesize

447

corrinoids de novo that can be used by corrinoid auxotrophs such as Dehalogenimonas

448

spp. Previous studies have shown that the metabolism of OHRB can be altered by

449

supplying different forms of corrinoids i.e. cyanocobalamin added exogenously to the

450

medium or corrinoids supplied by a methanogenic enrichment culture.28 Therefore, here

451

we aimed to investigate whether the presence of methanogens may exert an effect on the

452

isotopic fractionation of 1,2-DCP due to the corrinoid source or due to corrinoid

453

concentration. In addition, the results may help to identify a yet unperceived

454

concomitant cometabolic transformation of 1,2-DCP. This information is of importance

455

to interpret and increase confidence in the isotopic fractionation data collected from the

456

field since growth of OHRB is mostly favored in methanogenic zones. Therefore,


Page 21 of 35


457

carbon isotopic composition and concentration of 1,2-DCP was monitored in

458

methanogenic (fifth transfer) and non-methanogenic enrichment cultures (eleventh

459

transfer) containing 25 mM BES during 1,2-DCP dechlorination. In methanogenic

460

cultures, 94% of 1,2-DCP was transformed to propene within 13 days, whereas non-

461

methanogenic cultures were much slower and only 77% transformation was reached

462

after 21 days. In both methanogenic and non-methanogenic cultures, 1,2-DCP was

463

significantly enriched in

464

considered carbon isotopic compositions of spiked 1,2-DCP was -29.1 ± 0.5‰ and -

465

29.8 ± 0.3‰, respectively for the two experiments, and were calculated from the

466

average delta value obtained from aqueous control standards injected together with the

467

samples. These values did not differ significantly from each other (within their 1σ) nor

468

to the ones obtained in abiotic controls after the incubation period (-29.2 ± 0.2‰ and -

469

29.9 ± 0.1‰, respectively for each series). Therefore, the equilibrium of 1,2-DCP

470

between headspace and liquid in the serum bottles did not affect substantially the

471

measured 1,2-DCP delta value. However, in methanogenic cultures, the inoculum

472

contained a low concentration of residual 13C-enriched 1,2-DCP. This resulted in a more

473

13

474

2). In non-methanogenic cultures, no 1,2-DCP was detected in live controls without 1,2-

475

DCP, demonstrating that no enriched 1,2-DCP was transferred with the inoculum and

476

the -29.9 ± 0.1‰ value from abiotic controls was used as initial (δ13C0). Taking this into

477

account, 1,2-DCP carbon isotope fractionation (εCbulk) was calculated with the Rayleigh

478

equation (equation 2) for methanogenic (-15.3 ± 0.7‰) and non-methanogenic (-13.7 ±

479

2.0‰) enrichment cultures (Table 1). These values were statistically identical according

480

to their 95% confidence intervals indicating that the reaction mechanism was not

481

detectably affected by the presence/absence of methanogens in our consortium. Our

13

C during the transformation to propene (Figure 2). The

C-enriched 1,2-DCP initial value (δ13C0) of -27.5 ± 0.1‰ in this experiment (Figure



Page 22 of 35

482

results are in agreement with those reported recently in which trichloroethene

483

fractionation was not significantly different in two different Dehalococcoides-

484

containing enrichments cultures when methanogenic activity was either inhibited or

485

promoted.29

486 487

3.5. Calculation of carbon isotopic fractionation and AKIE values of 1,2-DCP

488

dechlorination

489

Combined εCbulk (-15.0 ± 0.7‰) was calculated by plotting methanogenic and non-

490

methanogenic data together (Figure 3) giving a correlation factor of 0.991 indicating

491

that dichloroelimation of 1,2-DCP to propene is well described by the Rayleigh model.

492

It is expected that species containing the same reductive dehalogenase produce similar

493

fractionation of 1,2-DCP, as previously observed for Dehalococcoides strains RC and

494

KS (-10.8 ± 0.9‰ and -11.3 ± 0.8‰, respectively).15 However, the obtained 1,2-DCP

495

εCbulk of -15.0 ± 0.7‰ for our Dehalogenimonas-containing culture differs significantly

496

according to 95% confidence intervals. Although this difference is intriguing, it is

497

known that species belonging to the same genus and harboring the same functional

498

dehalogenase can produce significantly different isotopic fractionation of chlorinated

499

compounds.30 This phenomenon is commonly attributed to physiological processes such

500

as transport of substrate across the cell membrane or differences in the active site of the

501

enzyme.

502

To distinguish if dichloroelimination proceeds via a stepwise or concerted mode

503

AKIE values were calculated (equation 4) using the combined carbon isotopic

504

fractionation obtained (Table 1). Assuming that the 1,2-DCP dichloroelimination

505

reaction was stepwise, involving the cleavage of one C-Cl bond in the transition state,

506

the combined εreactive value was -43 ± 2‰ and the corresponding AKIE 1.045 ± 0.002.


Page 23 of 35


507

Assuming that the reaction was concerted, involving the simultaneous cleavage of both

508

C-Cl bonds, the combined εreactive value was -22 ± 1‰ corresponding to an AKIE value

509

of 1.023 ± 0.001. Therefore, both AKIE values were lower than the theoretical

510

maximum carbon primary KIE (“semiclassical Streitwieser Limits”) for the complete

511

cleavage of a C-Cl bond (1.057).31 However, considering the indication of Elsner et al.

512

2005 that realistic values with transition states at about 50% bond cleavage can be

513

expected to be half as pronounced (AKIE = 1.03),11 the concerted reaction with an

514

obtained AKIE value lower than that (1.023) might be more probable in our case than

515

the stepwise (1.045), whereas for Fletcher et al. 2009 both values were still lower

516

enough (1.017 and 1.033, respectively).15

517

In addition, carbon isotopic composition of propene was measured in non-

518

methanogenic enrichment cultures to confirm the stoichiometric transformation from

519

1,2-DCP by isotopic mass balance. Equation 6 was used to calculate the newly

520

generated propene δ13C value. As shown in Fig. 2, 13C-depleted propene was generated

521

by 1,2-DCP dechlorination confirming isotope fractionation and it was getting

522

significantly enriched along the 1,2-DCP dichloroelimination (δ13Cpropene values from -

523

43.4‰ to -36.3‰). Moreover, by applying equation 5, the concentration-weighted

524

average δ13Csum was nearly constant at -31 ± 1‰ which is not statistically different from

525

the considered initial 1,2-DCP composition in this experiment (-29.8 ± 0.3‰),

526

suggesting a close carbon isotopic mass balance during dichloroelimination.

527

In summary, we established a stable Dehalogenimonas-containing culture that

528

exclusively dechlorinates vicinally chlorinated alkanes. Dechlorination of 1,2-DCP was

529

accompanied by a change in the isotope composition of 1,2-DCP. Production of

530

propene from 1,2-DCP was stoichiometric and also fits with the carbon isotopic

531

balance. The calculated εCbulk was in the same order of magnitude but differs from that



Page 24 of 35

532

reported for Dehalococcoides mccartyi strain RS and strain KC, although the dcpA gene

533

encoding 1,2-DCP reductive dehalogenase was also identified in our culture. The

534

carbon isotopic factor determined in this study can provide useful information to apply

535

compound specific stable isotope analysis for quantification of in situ dechlorination of

536

1,2-DCP in aquifers.

537 538

ACKNOWLEDGMENTS

539 540

This work has been funded by the Spanish Ministry of Economy and Competitiveness

541

(project CTM2013-48545-C2-1-R) and supported by the Generalitat de Catalunya

542

(Consolidated Research Group 2014-SGR-476). The Department of Chemical

543

Engineering of the Universitat Autònoma de Barcelona (UAB) is member of the Xarxa

544

de Referència en Biotecnologia de la Generalitat de Catalunya. E.M-U is indebted to the

545

UAB APOSTA grant that was critical for initiating this research. M.R. acknowledges

546

support from a Marie Curie Career Integration Grant in the framework of the IMOTEC-

547

BOX project (PCIG9-GA-2011-293808). S.H.M acknowledges support from the

548

Ministry of Education Malaysia (SLAI-UMP Scholarship) for a predoctoral fellowship

549

and L.A. acknowledges support by the DFG (FOR1530). We thank Dr. William M.

550

Moe for providing genomic DNA from Dehalogenimonas lykanthroporepellens BL-

551

DC-9.

552 553

SUPPORTING INFORMATION AVAILABLE

554

Additional tables and figures are included. This information is available free of charge

555

via the Internet at http://pubs.acs.org.

556


Page 25 of 35


557

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558 559

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2006. 72, 2765–2774; DOI 10.1128/AEM.72.4.2765-2774.2006.

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[27] Löffler, F. E., Yan, J., Ritalahti, K. M., Adrian, L., Edwards, E. A., Konstantinidis,

651

K. T., Müller, J. A., Fullerton, H., Zinder, S. H., Spormann, A. M. Dehalococcoides

652

mccartyi gen. nov., sp. nov., obligate organohalide-respiring anaerobic bacteria, relevant

653

to halogen cycling and bioremediation, belong to a novel bacterial class,

targeting

PCR

16S

rRNA

targeting

genes

of

the

Dehalogenimonas

16S

rRNA

and

organohalide-respiring

species

reductive

in

the

dehalogenase

genus

1,1,2,2-

genes


Page 29 of 35


654

Dehalococcoidetes classis nov., within the phylum Chloroflexi. Int J Syst Evol.

655

Microbiol. 2013, 63, 625-635; DOI 10.1099/ijs.0.034926-0.

656

[28] Johnson, D. R.; nemir, A.; Andersen, G. L.; Zinder, S. H.; Alvarez-Cohen, L.

657

Transcriptomic microarray analysis of corrinoid responsive genes in Dehalococcoides

658

ethenogenes

659

http://dx.doi.org/10.1111/j.1574-6968.2009.01569.x.

660

[29] Harding, K. C.; Lee, P. K.; Bill, M.; Buscheck, T. E.; Conrad, M. E.; Alvarez-

661

Cohen, L. Effects of varying growth conditions on stable carbon isotope fractionation of

662

trichloroethene (TCE) by tceA-containing Dehalococcoides mccartyi strains. Environ.

663

Sci. Technol. 2013, 47, 12342-12350; DOI 10.1021/es402617q.

664

[30] Marco-Urrea, E.; Nijenhuis, I.; Adrian, L. Transformation and carbon isotope

665

fractionation of tetra- and trichloroethene to trans-dichloroethene by Dehalococcoides

666

sp.

667

10.1021/es1023459.

668

[31] Huskey, W. P. Origins and interpretations of heavy-atom isotope effects. In

669

Enzyme Mechanism from Isotope Effects; Cook, P. F., Ed.; CRC Press: Boca Raton, FL

670

1991; pp 37.

strain

strain

CBDB1.

195.

FEMS

Environ.

Sci.

Microbiol

Technol.

Lett.

2009,

2009,

45,

198-206;

1555-1562;

DOI

DOI

671 672 673



674

Page 30 of 35

FIGURE LEGENDS

675 676

Figure 1. Increase of Dehalogenimonas 16S rRNA gene copy numbers in Besòs River

677

cultures after consuming different doses of 1,2-DCP. Panel A: Consumption of 1,2-DCP

678

over time. The culture received several additions of 1,2-DCP, as indicated by the

679

arrows. Panel B: Dehalogenimonas 16S rRNA gene copies per mL of DNA in the 1,2-

680

DCP amended cultures. Error bars represent the standard deviation of copy numbers

681

measured in duplicate experiments for each point injected in triplicate.

682 683

Figure 2. Carbon isotopic composition of 1,2-DCP (circles) in methanogenic (solid

684

symbols) and non-methanogenic enrichment cultures containing 25 mM BES (open

685

symbols) during 1,2-DCP dechlorination. In non-methanogenic enrichment cultures,

686

carbon isotopic composition of propene was measured (stars) and values were corrected

687

to represent the generated propene (triangles) following equation 6 taking into

688

consideration the initial transfer of residual propene with the inoculum (see text). The

689

dashed line indicates the expected carbon isotopic mass balance which corresponds to

690

the initial 1,2-DCP composition (average value of -29.8 ± 0.3‰, n =11). The error bars

691

showing the one standard deviation (1σ) for duplicate measurements are smaller than

692

the symbols.

693 694

Figure 3. Double logarithmic plot according to the Rayleigh equation of the carbon

695

isotope ratio versus the residual concentration of 1,2-DCP during dechlorination by

696

methanogenic (solid symbols) and non-methanogenic enrichment cultures containing 25

697

mM BES (open symbols). The solid line corresponds to a linear regression model for


Page 31 of 35


698

total combined data (check Table 1 for more details) and gray dashed lines to its

699

associated 95% confidential intervals. Data points show their related error bars.



700

Page 32 of 35

FIGURE 1

A

1,2-DCP (µmol)

200 150 100 50

16S rRNA gene copies mL-1

0 7e+8

B

6e+8 5e+8 4e+8 3e+8 2e+8 1e+8 0 0

701

5

10

15

20

25

30

Time (d)


Page 33 of 35


702

FIGURE 2

703 704 705 20 10

δ13C [‰VPDB]

0 -10 -20 -30 -40 -50 0.0

0.2

0.4 0.6 0.8 Degraded fraction of 1,2-DCP

1.0

706



707

Page 34 of 35

FIGURE 3

708 0.05

ln(Rt/R0)

0.04

0.03

0.02

0.01

0.00 -3.5

-2.5

-1.5 ln(Ct/C0)

-0.5

0.5

709


Page 35 of 35


710

TABLE LEGENDS

711

Table 1. Carbon Isotope Fractionation (εCbulk) with 95% confidence intervals (95% CI) and calculated AKIE values assuming either stepwise or

712

concerted reductive dechlorination of 1,2-DCP. AKIE valuec Experiment

Suspected degrader

ɛCbulk (‰)

Na

R2

Db (%)

Non-methanogenic culture RC

Dehalococcoides

-10.8 ± 0.9

7

>0.96

>90

1.033 ± 0.003 1.016 ± 0.001

[13]

Non-methanogenic culture KS

Dehalococcoides

-11.3 ± 0.8

7

>0.96

>90

1.033 ± 0.003 1.017 ± 0.001

[13]

Methanogenic culture BR

Dehalogenimonas

-15.3 ± 0.7

12

0.996

94

1.046 ± 0.002 1.023 ± 0.001

This study

Non-methanogenic culture BR

Dehalogenimonas

-13.7 ± 2.0

8

0.98

77

1.042 ± 0.007 1.021 ± 0.003

This study

BR total combined data

Dehalogenimonas

-15.0 ± 0.7

20

0.991

94

1.045 ± 0.002 1.023 ± 0.001

This study

Stepwise

Concerted

Reference

713

a

714

b

715

c

716

carbon atoms located at the reactive site and z is the number of indistinguishable reactive sites, correcting the effects of intramolecular isotopic

717

competition. For 1,2-DCP, n =3 and the (x,z) considered for stepwise (1,1) and for concerted (2,1).

N: number of data points analyzed in duplicate. D%: maximum percentage of 1,2-DCP degradation which could be analyzed by GC-IRMS.

AKIE values were calculated according to reference [10] being n the number of carbon atoms in the molecule of which x is the number of

35

ACS Paragon Plus Environment

Stable Carbon Isotope Fractionation During 1,2-Dichloropropane-to

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