Geochemical Parameters and Reductive Dechlorination Determine

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Geochemical Parameters and Reductive Dechlorination Determine Aerobic Cometabolic vs Aerobic Metabolic Vinyl Chloride Biodegradation at Oxic/anoxic Interface of Hyporheic Zones Siavash Atashgahi, Yue Lu, Javier Ramiro-Garcia, Peng Peng, Farai Maphosa, Detmer Sipkema, Winnie Dejonghe, Hauke Smidt, and Dirk Springael Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05041 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Page 1 of 25

Environmental Science & Technology

1

Geochemical Parameters and Reductive Dechlorination Determine

2

Aerobic

3

Biodegradation at Oxic/anoxic Interface of Hyporheic Zones

Cometabolic

vs

Aerobic

Metabolic

Vinyl

Chloride

4 5

Siavash Atashgahi,*,†,‡,§ Yue Lu,†,#$ Javier Ramiro-Garcia,†,# Peng Peng,† Farai Maphosa,†

6

Detmer Sipkema,† Winnie Dejonghe,‡ Hauke Smidt,† and Dirk Springael§

7 8 9 10

†

Laboratory of Microbiology, Wageningen University & Research, Stippeneng 4, 6708 WE

Wageningen, The Netherlands

11 12

‡

13

Technology, Boeretang 200, 2400 Mol, Belgium

Flemish Institute for Technological Research (VITO), Separation and Conversion

14 KU Leuven, Division of Soil and Water Management, Kasteelpark Arenberg 20, B-3001

15

§

16

Heverlee, Belgium

17 Corresponding Author

18

*

19

Phone: +31 317 484683; e-mail: [email protected]

20 21 22

#

Equal contribution

$

Current address: College of Environmental Science and Engineering, Hunan University,

Changsha, People’s Republic of China

23

1

ACS Paragon Plus Environment


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24

Abstract

25

Hyporheic zones mediate vinyl chloride (VC) biodegradation in groundwater discharging

26

into surface waters. At the oxic/anoxic interface (OAI) of hyporheic zones subjected to

27

redox oscillations, VC is degraded via co-existing aerobic ethenotrophic and anaerobic

28

reductive dechlorination pathways. However, the identity of aerobic VC degradation

29

pathways (cometabolic vs metabolic) and their interactions with reductive dechlorination

30

in relation to riverbed sediment geochemistry remain ill-defined. We addressed this using

31

microcosms

32

atmosphere. Under oxic atmosphere, aerobic metabolic VC oxidation was absent in

33

sediments with high total organic carbon (TOC) and VC was reductively dechlorinated to

34

ethene. Ethene was oxidized by ethenotrophs that can degrade VC cometabolically.

35

Contrastingly, VC was metabolically oxidized by ethenotrophs in low-TOC sediments with

36

low reductive dechlorination potential. Accordingly, enrichment and isolation of metabolic

37

VC-oxidizing ethenotrophs was successful only from the low-TOC sediment. Sequence

38

analysis of etnE genes from the microcosms as well phylogenetic typing of the isolates

39

showed that ethenotrophs in the sediments were facultative anaerobic Proteobacteria

40

capable of coping with OAI-associated redox fluctuations. Our results suggest that local

41

sediment heterogeneity supports/selects divergent VC degradation processes at the OAI

42

and that high reductive dechlorination potential suppresses development of aerobic

43

metabolic VC oxidation potential.

containing

OAI

sediments

incubated

under

2


fluctuating

oxic/anoxic

Page 3 of 25


44

INTRODUCTION

45

Chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) are

46

among the most frequently detected groundwater contaminants. At many industrial sites,

47

CE-contaminated groundwater discharges into surface water through the riverbed

48

hyporheic zone. The

49

groundwater and surface water with steep vertical gradients of various physicochemical

50

parameters such as organic matter content and redox potential.1 Hyporheic zones have

51

recently been recognized as biological filters capable of reducing or preventing CE

52

transfer from groundwater to surface water.2-10 The anoxic organic-rich sediment layers

53

can mediate partial or complete reductive dechlorination of CEs by organohalide-respiring

54

bacteria such as Dehalococcoides mccartyi (Dcm).2,6,11,12 On the other hand, the oxic top

55

sediment at the oxic/anoxic interface (OAI) provides conditions for complementary

56

aerobic cometabolic13-15 or growth-coupled16-20 degradation of cis-dichloroethene (cDCE)

57

and vinyl chloride (VC) derived from reductive dechlorination of parent compounds PCE

58

and TCE.

59

Cometabolic aerobic degradation of cDCE and VC is performed by aerobic bacteria

60

containing oxygenase enzymes with broad substrate range which fortuitously degrade

61

VC/cDCE using phenol, toluene, ethene, methane, propane or ammonia as the growth-

62

supporting

63

(ethenotrophs) is of particular interest for VC/cDCE degradation in hyporheic sediments

64

since ethene produced from reductive dechlorination of parent CEs can be used as a

65

primary substrate for cometabolic degradation of residual VC/cDCE in aerobic zones.

66

Moreover,

67

Nocardioides)23-25 and Proteobacteria (Pseudomonas, Ochrobactrum, Ralstonia)20,26-29,

68

have also been reported to mediate metabolic VC degradation allowing assimilation of VC

69

and

70

monooxygenase (EtnABCD) encoded by the etnABCD operon, yielding epoxyethane from

71

ethene and chlorooxirane from VC.21,23,24 These reactive epoxides are further degraded

hyporheic zone is the interstitial

substrate.13,21,22

ethenotrophs,

growth.

Ethene/VC

Ethene

including

degradation

members

degradation

is

by

of

initiated

sediment

zone between

ethene-assimilating

Actinobacteria

by

3


a

organisms

(Mycobacterium,

four-component

alkene


Page 4 of 25

72

by an epoxyalkane-coenzyme M transferase (EtnE) encoded by etnE.23,28 The etnE gene

73

is located on large linear plasmids in ethenotrophic members of Actinobacteria and

74

Proteobacteria29,30 and is used as a biomarker to study abundance31-33 and diversity32,33

75

of aerobic VC/ethene degraders. Upon extended exposure to VC in axenic cultures,

76

ethenotrophic strains of Mycobacterium25,34 and Pseudomonas27 were shown to transit

77

from cometabolic to growth-linked VC degradation. In Mycobacterium sp. strain JS623,

78

missense mutations in the etnE gene were proposed to mediate this transition.35 Further,

79

Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD grown in Luria-Bertani

80

broth without VC showed diminished EtnE activity and divergent etnE sequence

81

compared to corresponding VC-grown strains.28

82

In a recent study, we showed co-existence of pathways mediating anaerobic VC

83

respiration and aerobic VC/ethene degradation in the OAI sediments of the Belgian Zenne

84

River11 affected by a CE-contaminated groundwater plume.5 The observed biodegradation

85

pathways in sediment microcosms incubated under oxic/anoxic atmosphere were

86

dependent on the sediment characteristics i.e. total organic carbon (TOC) content and

87

sediment grain size. Under anoxic atmosphere, VC was stoichiometrically converted to

88

ethene by Dcm independent of the sediment characteristics. In contrast, under oxic

89

atmosphere, Dcm seemed protected from oxygen toxicity in sediments with high TOC

90

content and fine grain size and reductive dechlorination remained the dominant VC

91

removal

92

accumulation, most likely due to ethene degradation by ethenotrophs,11 some of which

93

can also degrade VC cometabolically while growing on ethene as the growth-supporting

94

substrate.21 In contrast, Dcm seemed inactive under oxic atmosphere in sediments with

95

low TOC content and coarse grain structure. Hence, the observed VC degradation was

96

likely mediated by ethenotrophs adapted to metabolic VC oxidation.11 Therefore, our

97

aims in this study were i) to distinguish between aerobic cometabolic vs aerobic

98

metabolic VC degradation in OAI sediments of hyporheic zones, and ii) to assess the role

99

of reductive dechlorination governed by local sediment heterogeneity in mediating

100

mechanism.

However,

VC

removal

was

not

cometabolic vs metabolic aerobic VC degradation. 4


accompanied

by

ethene

Page 5 of 25


101

To this end, we prepared similar sediment microcosms using the same high and low

102

organic sediment types as done before11 and incubated the microcosms under sequential

103

static/shaking conditions and oxic/anoxic atmosphere. We hypothesized that under oxic

104

shaking conditions reductive dechlorination would be suppressed by rigorous oxygen

105

exposure. Besides inhibiting reductive removal of VC, this would also prevent ethene

106

production and hence limit the availability of the growth-supporting substrate for

107

cometabolic oxic VC degradation.27,34,36 Therefore, under shaking oxic conditions, only

108

cultures capable of metabolic VC oxidation would be able to grow on VC. In addition, we

109

applied the Illumina MiSeq platform to sequence a fragment of the etnE gene that

110

contains previously reported missense mutations suggested to mediate metabolic VC

111

degradation35 and attempted enrichment and isolation of VC-assimilating bacteria from

112

both sediments. Finally, the sequential incubation enabled us to examine how redox

113

fluctuations occurring in the OAI of hyporheic zones5 affect resistance and resilience of

114

aerobic and anaerobic VC-degrading microbial guilds and hence VC fate.

115 116

Materials and Methods

117

Site material

118

Sediment samples used in this study were taken from the Zenne River in Machelen-

119

Vilvoorde, Belgium. A groundwater plume mainly contaminated with cDCE and VC is

120

flowing from the bank aquifer into the River Zenne. A detailed description of the test site

121

was provided elsewhere.5 River-bed sediment samples up to a depth of 20 cm were

122

collected on May 2010 from two locations in the riverbed. The sediment at location P26 is

123

characterized by a high total organic carbon content (TOC; 0.73% (w/w)) and fine grain

124

sediment structure, whereas at location P25 low TOC (0.31% (w/w)) and coarse

125

sediment structure are found. Sediment samples were collected in air-tight glass jars and

126

transported to the laboratory in a cooler. Surface water samples were collected as grab

127

samples in glass bottles, without leaving a headspace and used for microcosm

128

preparation upon arrival in the lab. The characteristics of sediment and surface water

129

used in this study were reported previously.11 5



130 131

Microcosm set-up and reversibility test

132

Sediment microcosms were prepared from P26 or P25 materials as described earlier.11

133

VC was always amended at aqueous concentration of 70 μM (6 µmol/bottle) and its

134

biodegradation was followed according to the sequential treatments denominated as ‘a’

135

to ‘g’ as outlined in Figure 1, i.e., implementation of oxic or anoxic headspace under

136

static or shaken conditions. Unless stated otherwise, each treatment included three

137

consecutive VC amendments. Each time when VC was depleted, the headspace of the

138

microcosms was flushed three cycles (15 min each with one hour intervals) with oxygen

139

free nitrogen, before VC and oxygen (when necessary) were replenished. To convert the

140

headspace from anoxic to oxic, sterile oxygen gas (ultra-pure, Air Products, Belgium;

141

filter sterilized over a 0.2-µm filter) was injected at an initial concentration of 7%

142

(vol/vol) of the nitrogen headspace after the withdrawal of an equal volume of nitrogen

143

headspace. Oxygen was replenished during oxic incubations when concentrations in the

144

headspace were below 5% (vol/vol) in order to keep a semi-constant headspace

145

concentration. Shaking was achieved by incubating the microcosms horizontally on a

146

rotary shaker (120 rpm). All incubations were performed at 22 °C. Unless stated

147

otherwise, at the end of each treatment, slurry samples (~2 g, after thorough mixing)

148

were taken for molecular analysis. This was followed by non-sterile surface water

149

addition to keep a constant volume and oxic/anoxic headspace adjustment according to

150

each oxic/anoxic treatment.

151 152

Enrichment and isolation of aerobic metabolic VC oxidizing bacteria

153

At the end of the third VC spike under oxic static incubation, 7 mL slurry from replicate

154

P25 and P26 microcosms was transferred to 160 mL vials containing 63 mL mineral salts

155

medium (MSM)18 for enrichment of aerobic metabolic VC-oxidizing bacteria. The cultures

156

were amended with 70 µM VC and sterile oxygen (7 % headspace) as described above

157

and incubated static at room temperature. In cultures that depleted VC, 70 µM VC and

158

oxygen (if necessary) were re-amended and degradation was followed for two more 6


Page 6 of 25

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159

consecutive VC spikes. After degradation of the third VC spike, 2 ml samples were taken

160

from the suspension for DNA extraction. Subsequently, cultures that showed VC

161

degrading activity were transferred to fresh MSM and aerobic VC (70 µM) degradation

162

was followed for five transfers. From those highly enriched cultures, 10-1-10-5 dilutions

163

were spread onto MSM agar plates and incubated in a desiccator with a 1% (v/v) VC-air

164

atmosphere. After two months, individual colonies were aseptically streaked on new MSM

165

agar and identically re-incubated in the presence of 1% VC or absence of VC as control.

166

Colonies that only showed growth under VC atmosphere were aseptically transferred to

167

bottles containing MSM with 70 µM VC as the sole carbon source and sterile oxygen

168

(100% headspace) and incubated static. Each time when VC was degraded, the

169

concentration of VC was increased step-wise with each new amendment to a final

170

concentration of 6 mM. At the end of the last VC spike, 2 ml culture samples were taken

171

for DNA extraction. Identification of strains was done by partial sequencing of the

172

amplified 16S rRNA gene as described.25

173 174

DNA extraction and quantitative PCR (qPCR)

175

DNA was extracted from slurry samples as described.37 qPCR analyses targeting the Dcm

176

16S rRNA gene and bvcA, vcrA, etnC and etnE catabolic genes were performed as

177

reported.11 The results of qPCR analyses were normalized by dividing the number of gene

178

copies at any time point to the gene copy number at time zero of a particular treatment

179

and presented as fold change. For the time zero values of Dcm 16S rRNA gene and bvcA,

180

vcrA that were below the detection limit of 10 3 copies/g sediment, the detection limit was

181

used as the denominator for normalization.

182 183

etnE MiSeq sequencing

184

A total of 20 samples was selected for etnE amplicon sequencing including time zero

185

sediment samples from both locations P25 and P26 as well as samples from the duplicate

186

microcosms after selected treatment steps including oxic static, anoxic static-2, anoxic

187

shaking, and oxic shaking steps. A 284 bp fragment of the etnE gene that includes the 7



188

missense mutations reported to be involved in the transition from ethene- to VC-

189

assimilation (i.e. use of VC as a growth-supporting substrate)35 was amplified using a

190

newly designed forward primer, CoM-F663 (Table S2), in combination with the CoM-R2E

191

reverse primer.31 Barcoded etnE gene amplicons were generated using a 2-step PCR

192

strategy as outlined in supporting information.

193

Demultiplexing, OTU generation, sequencing error correction and chimera checking were

194

done using NG-Tax,38 using default parameters with no taxonomic assignment. To

195

minimize the presence of sequencing errors, the read length was set to 100 nucleotides

196

for both paired-end reads. Translation to amino acids (aa) yielded a sequence of 32 aa

197

for the forward read, a sequence of 33 aa for the reverse reads leaving a gap of nine aa

198

between both sequence reads.

199 200

Cloning and sequencing of etnE amplicons

201

891 bp etnE fragments were obtained from aerobic metabolic VC-oxidizing enrichment

202

cultures and isolates originating from the microcosms. The etnE gene was amplified as

203

described23 using GoTaq DNA Polymerase Kit (Promega, Leiden, The Netherlands) and

204

primers CoM-F1L and CoM-R2E.30 The PCR products from the enrichment cultures were

205

cloned as described,39 and together with the purified PCR products from the isolates were

206

Sanger sequenced (GATC-Biotech, Konstanz, Germany) with the CoM-F1L primer.

207 208

Chemical analyses

209

Concentrations of VC and ethene were determined via head-space analysis on a Varian

210

GC-FID (CP-3800) and the total content per microcosm bottle calculated as described.11

211

Headspace oxygen content was determined by injecting 100-μL headspace samples into

212

a Hewlett-Packard (HP) 6890N gas chromatograph equipped with an HP-Plot MoleSieve

213

column (15 m × 0.53 mm; film thickness 25 μm nominal) and a thermal conductivity

214

detector. Helium (6 mL/min) was the carrier gas, and the injector (split at 10:1), oven,

215

and detector were maintained at 90, 40, and 150 °C, respectively. The detection limit for

216

oxygen was ca. 0.1 mg/L of aqueous phase. 8


Page 8 of 25

Page 9 of 25


217 218

Sequence data

219

Nucleotide sequences of the PCR-amplified etnE gene fragments obtained by MiSeq

220

sequencing are available at the European Bioinformatics Institute under accession

221

number PRJEB14318. The etnE sequences obtained from aerobic metabolic VC-oxidizing

222

enrichment cultures and isolates were deposited in GenBank under accession numbers

223

KX359820 to KX359843 (See Table S1).

224 225

Results

226

VC degradation and dynamics of molecular biomarkers in sediment microcosms

227

under periodic redox oscillation

228

VC was stoichiometrically dechlorinated to ethene during the first three VC amendments

229

under anoxic conditions (anoxic static-1, treatment a) in microcosms prepared from

230

either P26 or P25 sediments (Figure 1A&C, treatment a). VC dechlorination was

231

accompanied by a more than 15-fold increase of the abundance of the Dcm 16S rRNA,

232

vcrA and bvcA genes (Figure 1B&D, treatment a) compared to their respective time zero

233

results (Figure S1, t0). In contrast, the abundance of the etnC gene decreased three-

234

and 15-fold in the P26 and P25 microcosms, respectively (Figure 1B&D, treatment a)

235

relative to time zero results (Figure S1, t0), whereas the etnE gene abundance remained

236

rather stable. In the subsequent treatment under oxic atmosphere (oxic static, treatment

237

b), VC disappearance was noted in both microcosms containing P26 and P25 sediment

238

with concomitant ethene accumulation in P26 microcosms (Figure 1A, treatment b).

239

There was concurrent four- and 15-fold increase in etnC abundance in both P26 and P25

240

microcosms (Figure 1B&D, treatment b) and a two-fold increase in etnE abundance in

241

P26 microcosms relative to anoxic static-1 treatment (Figure 1B, treatment a). In

242

contrast, the abundance of reductive dechlorination associated biomarkers, i.e. Dcm 16S

243

rRNA, vcrA and bvcA genes dropped more than a 15-fold in P25 microcosms while they

244

were rather stable in P26 microcosms (Figure 1B&D, treatment b) relative to anoxic

245

static-1 treatment (Figure 1B&D, treatment a). 9



Page 10 of 25

246

The microcosms were subsequently converted back to anoxic conditions (anoxic static-2,

247

treatment c). This resulted in resumed ethene formation in both P26 and P25 microcosms

248

(Figure 1A&C, treatment c) as well as a more than 25-fold increased abundance of the

249

Dcm 16S rRNA, vcrA and bvcA genes in P25 microcosms (Figure 1D, treatment c)

250

compared to oxic static treatment (Figure D, treatment b). However, recovery of

251

reductive dechlorination was faster in P26 microcosms and took 84 days for removal of

252

three consecutive VC additions as opposed to 149 days for P25 microcosms (Figure

253

1A&C, treatment c).

254

Before incubation under shaking oxic conditions and to exclude a potential impact of

255

shaking on VC removal and associated microbiota also in the absence of oxygen, the

256

effect of shaking was first tested under anoxic conditions (anoxic shaking, treatment d).

257

In contrast to the stable abundance of etnE and etnC genes, shaking decreased the

258

abundance of the Dcm population three- and five-fold in P26 and P25 microcosms,

259

respectively (Figure 1B&D, treatment d) relative to anoxic static-2 treatment (Figure

260

1B&D, treatment c) and decreased VC reductive dechlorination rates considerably

261

especially in the P25 microcosms (Table S3) although complete dechlorination of VC was

262

still achieved (Figure 1C, treatment d). Subsequent anoxic static conditions (treatment e)

263

resulted in recovery of Dcm and VC reduction rates (Table S3), alleviating the negative

264

impact of shaking (Figure 1B&D, treatment e).

265

Subsequent shaking in the presence of an oxic atmosphere (treatment f) resulted in

266

rapid VC removal with no ethene detection in the P25 microcosms (Figure 1C, treatment

267

f). Accordingly, the abundance of the Dcm 16S rRNA, bvcA and vcrA genes decreased

268

more than a 200-fold and the abundance of the etnC and etnE genes increased 10- and

269

five-fold, respectively (Figure 1D, treatment f) compared to anoxic static-3 treatment

270

(Figure 1D, treatment e) indicating growth of ethenotrophs. Similarly, abundance of the

271

Dcm biomarkers declined in P26 microcosms (Figure 1B, treatment e) and reductive

272

dechlorination was suppressed, however, there was slow and limited VC removal (Figure

273

1A, treatment f) with no appreciable increase in etnC and etnE gene abundance (Figure

274

1B, treatment f) compared to anoxic static-3 treatment (Figure 1B, treatment e). When 10


Page 11 of 25


275

the oxic headspace was converted back to anoxic and static conditions (anoxic static-4,

276

treatment g), Dcm revival and reductive dechlorination of VC was noted only in P26

277

microcosms (Figure 1A&B, treatment g). In contrast, in P25 microcosms no VC removal

278

(Figure 1C, treatment g) or growth of Dcm (Figure 1D, treatment g) occurred even

279

though the oxic shaking period had been 32 days shorter than for P26 microcosms.

280 281

etnE sequences obtained from microcosms and original sediment by MiSeq

282

analysis

283

Our results imply that only the ethenotrophs residing in P25 are capable of aerobic

284

metabolic VC oxidation. To acquire support for this, we examined the sequence of etnE

285

gene amplicons derived from the original time zero sediment samples taken at P25 and

286

P26 locations and also selected samples from duplicate microcosms of P26 and P25

287

during different treatment periods. Previously, adaptation from co-metabolic to metabolic

288

VC degradation in actinobacterial ethenotrophs was shown to be linked with specific aa

289

changes in EtnE35 and hence the etnE gene provides a potential marker for co-metabolic

290

versus metabolic aerobic VC degradation. The deduced aa sequences from the three

291

predominant etnE OTUs recovered from the samples by MiSeq sequencing (OTU_10,

292

OTU_5, and OTU_15, representing 83 ± 13% of all observed etnE sequences) showed

293

88-89% aa similarity to EtnE of Mycobacterium strain JS623 and 97-100% aa similarity

294

to EtnE of Pseudomonas putida strain AJ28 and Ochrobactrum sp. strain TD28 over the

295

studied 65 aa length (Figure 2), indicating presence of proteobacterial rather than

296

actinobacterial ethenotrophs. The missense mutations in EtnE proposed to facilitate

297

aerobic metabolic VC oxidation in the ethenotroph Mycobacterium strain JS623 include a

298

W to G change at aa position 243 (W243G) and an R to L change at aa position 257

299

(R257L) (see Figure 2).35 However, these positions were conserved in the deduced EtnE

300

aa sequences from strains AJ and TD and those of OTU_10, OTU_5, and OTU_15 (Figure

301

2) and the remaining other recovered minor OTUs (data not shown). The time zero

302

samples of both locations were highly dominated by OTU_10 (Figure 3, Table S4) with

303

98.5% and 100% aa identities to those of TD and AJ strains, respectively, showing no 11



Page 12 of 25

304

sequence divergence between P25 and P26 locations. Moreover, OTU_10 was the

305

dominant etnE OTU in most samples obtained from P26 microcosms and in some P25

306

microcosm samples (Figure 3, Table S4). A striking difference between the samples

307

obtained from the P26 microcosms and some of the P25 microcosms was the

308

accumulation of OTU_5 over time in P25 sediments. The corresponding EtnE aa sequence

309

contains a mutation right after aa 257, i.e., A258E (Figure 2). However, OTU_5 was not

310

enriched in all P25 replicate microcosms and was for instance missing in samples taken

311

from one of the P25 microcosms after oxic shaking conditions which was enriched in

312

OTU_10 rather than OTU_5 (Figure 3, Table S4). Additional sequencing of etnE amplicons

313

from two independent DNA extracts from samples taken after oxic shaking condition

314

(treatment f) confirmed the lack of OTU_5 enrichment (Table S4), suggesting that OTU_5

315

cannot (solely) be responsible for the observed metabolic VC oxidation activity.

316

Moreover, the highest relative abundance of OTU_5 was found in samples withdrawn

317

from anoxic shaking conditions in P25 microcosms (Figure 3, Table S4).

318 319

Aerobic VC degradation by enrichment cultures and isolates and cloning and

320

sequencing of etnE gene

321

To further relate the occurrence of aerobic metabolic versus co-metabolic VC degradation

322

in the two different locations, we attempted to enrich metabolic VC degraders from P26

323

and P25 microcosms. However, no VC removal or ethene accumulation was noted in P26

324

enrichments even after 300 days of incubation (Figure S2). In contrast, the P25

325

enrichments degraded three consecutive spikes of 70 µM VC within 30 days without

326

accumulation of ethene (Figure S2).

327

Pseudomonas sp. and one Ochrobactrum sp. isolate were obtained (Figure S3) all

328

capable of aerobic growth with VC as the sole carbon and energy source (Figure S4).

329

Cloning and sequencing of etnE from the aerobic metabolic VC-oxidizing cultures

330

obtained from P25 microcosms, yielded 24 longer 891 bp etnE sequences, i.e., 20 from

331

the P25 enrichment cultures and four sequences from the bacterial isolates. Analysis of

332

these longer etnE sequences also showed conserved W243 and R257 positions in the

From the P25 enrichment cultures,

12


three

Page 13 of 25


333

deduced aa sequences (Figure 2). Over the 65 aa length studied by MiSeq, the longer

334

891 bp etnE sequences obtained from metabolic VC-oxidizing enrichments and isolates

335

showed 97-100 % aa similarity to the dominant OTU_10, OTU_5, and OTU_15 and also

336

to the corresponding EtnE region of strains AJ and TD (Figure 2). The 891 bp etnE

337

sequences further facilitated comparison to the EtnE variations reported in AJ and TD

338

strains28 as most of the EtnE variations in the latter strains fall outside the fragment

339

covered by the primers that were used for MiSeq sequencing (aa 1-229). Compared to

340

the EtnE obtained from VC-grown strains, EtnE of cells of AJ and TD strains grown in

341

Luria-Bertani broth was reported to differ at four and five aa positions, respectively. 28 We

342

compared the variation of that region (aa 1-229) between EtnE of strains JS623, AJ, and

343

TD and those obtained from the P25 enrichment cultures (20 sequences) and isolates

344

(four sequences) (Table S5). The EtnE aa sequences from the P25 enrichment cultures

345

and isolates showed 99.5% similarity to each other, 98.3% to the EtnE of VC-grown

346

strains AJ and TD and 92.3% to that of strain JS623. Compared to the consensus

347

sequence (Table S5), six EtnE aa sequences obtained from the enrichment cultures

348

showed a single random mutation per sequence indicating that those variations are not

349

likely to mediate aerobic metabolic VC oxidation. Furthermore, the positions of EtnE

350

variations in AJ and TD strains reported between cultures grown on VC or Luria-Bertani

351

broth without VC28 were not manifested in the EtnE aa sequences obtained from the

352

metabolic VC-oxidizing enrichment cultures and isolates, excluding a role for such a

353

mutation to mediate transition from ethene to VC as a growth-supporting substrate in the

354

cultures studied here.

355 356

Discussion

357

VC-contaminated groundwater passes through hyporheic zones before discharging into

358

surface water. Particularly in the OAI of hyporheic zones where VC-contaminated

359

groundwater encounters oxygen and where steep redox gradients and high microbial load

360

exist,40 the conditions are appropriate for a blend of anaerobic and aerobic VC

361

degradation.41 However, the nature of aerobic VC degradation (cometabolic vs metabolic) 13



Page 14 of 25

362

and interactions and potential synergies with reductive dechlorination in relation to the

363

sediment geochemistry in the OAI of hyporheic zones remain ill-defined.

364

The oxic shaking conditions were expected to disrupt sediment stratification and facilitate

365

diffusion of oxygen into the sediment matrix.42 This would stop the activity of oxygen

366

sensitive Dcm,43-45 eliminate interference of reductive dechlorination and provide

367

conditions to distinguish cometabolic from metabolic aerobic VC degradation. To this end,

368

we first controlled the impact of shaking under anoxic atmosphere on reductive

369

dechlorination to avoid artefacts due to shaking. Although shaking affected reductive

370

dechlorination rate (Table S3), no inhibition of the overall process was observed

371

(treatment d, Figure 1). Moreover, recovery under subsequent anoxic static incubation

372

was observed (treatment e, Figure 1). Mechanical disturbance due to vigorous shaking

373

may disrupt the integrity of sediment particle-associated biofilms leading to an increased

374

exposure to VC.

375

Implementing an oxic atmosphere under shaking conditions affected VC degradation in

376

the P25 and P26 microcosms differently (treatment b, Figure 1). The high VC degradation

377

rates (Table S3) concomitant with the growth of ethenotrophs despite the suppressed

378

reductive dechlorination and hence lack of ethene as a co-substrate for cometabolic VC

379

degradation (Figure 1C&D, treatment f) support the occurrence of aerobic metabolic VC

380

oxidation in the OAI of P25 sediments. In contrast, P26 microcosms were characterized

381

by inhibited reductive dechlorination, lack of detectable ethene accumulation and slow

382

and limited VC disappearance which was clearly linear (Figure 1A, treatment f) with no

383

appreciable increase in etnC and etnE gene copy numbers, indicating cometabolic

384

ethenotrophic rather than metabolic VC degradation (Figure 1B, treatment f). This

385

supports the hypothesis that under oxic static conditions in P26 sediment (Figure 1A,

386

treatment b), VC was mainly reductively dechlorinated to ethene by Dcm protected from

387

oxygen toxicity. In line with this, no enrichment culture capable of aerobic VC

388

degradation was obtained from P26 when a 10% v/v slurry was transferred to MSM

389

medium at the end of oxic static conditions (Figure S2). However, ethenotrophs present

390

in the OAI sediment of the P26 location might have contributed to VC disappearance by 14


Page 15 of 25


391

cometabolic VC degradation.27,34,36 Therefore, differences exist between ethenotrophic

392

processes in sediments of P26 and P25 with only ethenotrophs residing in P25 being

393

capable of aerobic metabolic VC oxidation.

394

Previously, it was shown that transition from cometabolic to metabolic VC degradation in

395

Mycobacterium JS623 was due to minor sequence divergences in the etnE gene.

396

Nevertheless, data from the etnE MiSeq amplicon sequencing as well as Sanger

397

sequencing of etnE from aerobic metabolic VC-oxidising enrichment and pure cultures

398

obtained from our study site do not agree with this. Instead, the sequencing and isolation

399

results suggest the presence of facultative anaerobic proteobacterial ethenotrophs

400

(Pseudomonas

401

(Mycobacterium and Nocardioides). Interestingly, this is in contrast to former reports on

402

etnE

403

enrichment cultures46 where the majority of the retrieved sequences were related to

404

Mycobacterium and Nocardioides. Similarly, stable isotope probing using

405

aerobic enrichment cultures obtained from VC-contaminated suboxic groundwater

406

identified Nocardioides as the main player for the labelled carbon uptake whereas

407

members of Pseudomonas were either absent32 or present as a minor fraction (~1%).46

408

As such, groundwater ecosystems appear better habitats for the strictly aerobic

409

actinobacterial ethenotrophs that are capable of metabolic VC oxidation at extremely low

410

oxygen concentrations (0.03 to 0.3 mg/L for pure cultures)25 found in shallow

411

aquifers47,48 and that were even isolated from nominally anoxic groundwater microcosms

412

possibly due to oxygen penetration during sampling.19 In contrast, the compact sediment

413

matrix and high organic carbon load of hyporheic zones likely support proteobacterial

414

ethenotrophs. In line with this, strains AJ and TD were isolated from enrichment cultures

415

obtained from a CE-contaminated lagoon and landfill, respectively, both representing

416

habitats that are known to be influenced by high organic carbon concentrations and

417

fluctuating redox conditions.29 Further, the redox conditions change frequently in the OAI

418

of hyporheic zones due to changing river and/or groundwater levels for instance after

419

precipitation events,5,6 which is congruent with the facultative anaerobic lifestyle of

diversity

and

from

Ochrobactrum)

(sub)oxic

rather

than

groundwaters33

strictly

and

aerobic

Actinobacteria

groundwater-derived

15


aerobic

13

C VC in


Page 16 of 25

420

proteobacterial ethenotrophs. In line with this, we noted resilience of ethenotrophs to

421

long-term incubation under strictly anoxic conditions in our microcosm.

422

Our sequencing results do not exclude the possibility of adaptation to metabolic VC

423

oxidation, and as hypothesized by others, mutations in downstream catabolic enzymes,

424

coenzyme M biosynthesis genes, or the plasmid copy number are possible contributors to

425

VC adaptation21,31. In fact, our former field measurements of VC concentrations are in

426

favour of such an evolution. Up to 2600 μg/L VC was documented to reach the top 20 cm

427

riverbed sediment layer at location P255 where the site properties such as low TOC11 are

428

not favorable for reductive dechlorination. This shows that relatively high loads of VC

429

were entering the OAI at this location that would favor adaptation to VC as growth-

430

supporting substrate by extended exposure of ethenotrophs to VC.27,34,35 At P26 on the

431

other hand, negligible amounts of VC (up to 81 μg/L) reached the top sediment layer5

432

due to high TOC and high reductive dechlorination activity,11 limiting exposure of

433

ethenotrophs to VC.

434

After rigorous shaking of microcosms under oxic atmosphere (treatment g, Figure 1), the

435

VC-respiring Dcm showed resistance and resilience to oxygen exposure only in sediments

436

of P26 (Figure 1A&B, treatment g). This is likely due to the high TOC and fine grain

437

structure of the sediment at this location that better protected Dcm11 in line with the fact

438

that known Dcm strains lose dechlorination activity irreversibly once exposed to air in

439

pure cultures.43,45 Members of Dcm have also shown limited resistance and resilience to

440

oxygen exposure in sediment free enrichment cultures, in particular VC-respiring strains

441

that are more susceptible to oxygen inhibition.44 This persistent reductive dechlorination

442

potential in P26 sediments and hence diminished VC concentration reaching OAI likely

443

precluded development of aerobic metabolic VC oxidation potential in P26 sediment

444

implying that prolonged exposure to VC is a prerequisite for evolution of aerobic

445

metabolic VC oxidation potential in hyporheic sediments.

446 447

Supporting information

16


Page 17 of 25


448

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

449

DOI: XXXX.

450

Figures showing qPCR results (Figure S1); VC fate in enrichment cultures (Figure S2);

451

phylogenetic tree of isolates (Figure S3); VC degradation by isolates (Figure S4). Tables

452

showing the etnE sequences used for primer design (Table S1); MiSeq primers, Unitags

453

and thermal cycling conditions (Table S2); first order VC degradation rates (Table S3);

454

the most abundant etnE OTUs (Table S4); variations in EtnE sequences (Table S5).

455 456

Notes

457

The authors declare no competing financial interest.

458 459

Acknowledgements

460

This study was supported by a VITO/KU Leuven PhD scholarship (EU FP7 project

461

AQUAREHAB, grant 226565) to S. Atashgahi. Furthermore, S. Atashgahi and H. Smidt

462

received support by a grant of BE-Basic-FES funds from the Dutch Ministry of Economic

463

Affairs and D. Springael by the Inter-University Attraction Pole (IUAP) “µ-manager” of the

464

Belgian Science Policy (BELSPO, P7/25). We thank C. Gielen, M. Maesen and Q. Simons

465

for laboratory technical support and acknowledge the China Scholarship Council for the

466

support to Y. Lu and P. Peng.

467 468

Figure legends

469

Figure 1. VC degradation and accumulation of ethene (A, C) and fold changes in Dcm

470

16S rRNA gene and bvcA, vcrA, etnC and etnE gene abundance compared to their

471

respective numbers at time zero (B, D) in VC-spiked microcosms of P26 (A, B) and P25

472

(C, D). Data presented in graphs A and C are the average of duplicate microcosms at

473

each time point with

Geochemical Parameters and Reductive Dechlorination Determine

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