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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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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
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Abstract
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Hyporheic zones mediate vinyl chloride (VC) biodegradation in groundwater discharging
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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
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metabolic VC oxidation potential.
containing
OAI
sediments
incubated
under
2
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fluctuating
oxic/anoxic
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INTRODUCTION
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Chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) are
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among the most frequently detected groundwater contaminants. At many industrial sites,
47
CE-contaminated groundwater discharges into surface water through the riverbed
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hyporheic zone. The
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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
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recently been recognized as biological filters capable of reducing or preventing CE
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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
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sediment at the oxic/anoxic interface (OAI) provides conditions for complementary
56
aerobic cometabolic13-15 or growth-coupled16-20 degradation of cis-dichloroethene (cDCE)
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and vinyl chloride (VC) derived from reductive dechlorination of parent compounds PCE
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and TCE.
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Cometabolic aerobic degradation of cDCE and VC is performed by aerobic bacteria
60
containing oxygenase enzymes with broad substrate range which fortuitously degrade
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VC/cDCE using phenol, toluene, ethene, methane, propane or ammonia as the growth-
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supporting
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(ethenotrophs) is of particular interest for VC/cDCE degradation in hyporheic sediments
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since ethene produced from reductive dechlorination of parent CEs can be used as a
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primary substrate for cometabolic degradation of residual VC/cDCE in aerobic zones.
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Moreover,
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Nocardioides)23-25 and Proteobacteria (Pseudomonas, Ochrobactrum, Ralstonia)20,26-29,
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have also been reported to mediate metabolic VC degradation allowing assimilation of VC
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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
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a
organisms
(Mycobacterium,
four-component
alkene
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by an epoxyalkane-coenzyme M transferase (EtnE) encoded by etnE.23,28 The etnE gene
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is located on large linear plasmids in ethenotrophic members of Actinobacteria and
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Proteobacteria29,30 and is used as a biomarker to study abundance31-33 and diversity32,33
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of aerobic VC/ethene degraders. Upon extended exposure to VC in axenic cultures,
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ethenotrophic strains of Mycobacterium25,34 and Pseudomonas27 were shown to transit
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from cometabolic to growth-linked VC degradation. In Mycobacterium sp. strain JS623,
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missense mutations in the etnE gene were proposed to mediate this transition.35 Further,
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Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD grown in Luria-Bertani
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broth without VC showed diminished EtnE activity and divergent etnE sequence
81
compared to corresponding VC-grown strains.28
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In a recent study, we showed co-existence of pathways mediating anaerobic VC
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respiration and aerobic VC/ethene degradation in the OAI sediments of the Belgian Zenne
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River11 affected by a CE-contaminated groundwater plume.5 The observed biodegradation
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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
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atmosphere, Dcm seemed protected from oxygen toxicity in sediments with high TOC
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content and fine grain size and reductive dechlorination remained the dominant VC
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removal
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accumulation, most likely due to ethene degradation by ethenotrophs,11 some of which
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can also degrade VC cometabolically while growing on ethene as the growth-supporting
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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
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likely mediated by ethenotrophs adapted to metabolic VC oxidation.11 Therefore, our
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aims in this study were i) to distinguish between aerobic cometabolic vs aerobic
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metabolic VC degradation in OAI sediments of hyporheic zones, and ii) to assess the role
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of reductive dechlorination governed by local sediment heterogeneity in mediating
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mechanism.
However,
VC
removal
was
not
cometabolic vs metabolic aerobic VC degradation. 4
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by
ethene
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To this end, we prepared similar sediment microcosms using the same high and low
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organic sediment types as done before11 and incubated the microcosms under sequential
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static/shaking conditions and oxic/anoxic atmosphere. We hypothesized that under oxic
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shaking conditions reductive dechlorination would be suppressed by rigorous oxygen
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exposure. Besides inhibiting reductive removal of VC, this would also prevent ethene
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production and hence limit the availability of the growth-supporting substrate for
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cometabolic oxic VC degradation.27,34,36 Therefore, under shaking oxic conditions, only
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cultures capable of metabolic VC oxidation would be able to grow on VC. In addition, we
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applied the Illumina MiSeq platform to sequence a fragment of the etnE gene that
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contains previously reported missense mutations suggested to mediate metabolic VC
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degradation35 and attempted enrichment and isolation of VC-assimilating bacteria from
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both sediments. Finally, the sequential incubation enabled us to examine how redox
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fluctuations occurring in the OAI of hyporheic zones5 affect resistance and resilience of
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aerobic and anaerobic VC-degrading microbial guilds and hence VC fate.
115 116
Materials and Methods
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Site material
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Sediment samples used in this study were taken from the Zenne River in Machelen-
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Vilvoorde, Belgium. A groundwater plume mainly contaminated with cDCE and VC is
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flowing from the bank aquifer into the River Zenne. A detailed description of the test site
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was provided elsewhere.5 River-bed sediment samples up to a depth of 20 cm were
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collected on May 2010 from two locations in the riverbed. The sediment at location P26 is
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characterized by a high total organic carbon content (TOC; 0.73% (w/w)) and fine grain
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sediment structure, whereas at location P25 low TOC (0.31% (w/w)) and coarse
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sediment structure are found. Sediment samples were collected in air-tight glass jars and
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transported to the laboratory in a cooler. Surface water samples were collected as grab
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samples in glass bottles, without leaving a headspace and used for microcosm
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preparation upon arrival in the lab. The characteristics of sediment and surface water
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used in this study were reported previously.11 5
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Microcosm set-up and reversibility test
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Sediment microcosms were prepared from P26 or P25 materials as described earlier.11
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VC was always amended at aqueous concentration of 70 μM (6 µmol/bottle) and its
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biodegradation was followed according to the sequential treatments denominated as ‘a’
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to ‘g’ as outlined in Figure 1, i.e., implementation of oxic or anoxic headspace under
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static or shaken conditions. Unless stated otherwise, each treatment included three
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consecutive VC amendments. Each time when VC was depleted, the headspace of the
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microcosms was flushed three cycles (15 min each with one hour intervals) with oxygen
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free nitrogen, before VC and oxygen (when necessary) were replenished. To convert the
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headspace from anoxic to oxic, sterile oxygen gas (ultra-pure, Air Products, Belgium;
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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
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headspace. Oxygen was replenished during oxic incubations when concentrations in the
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headspace were below 5% (vol/vol) in order to keep a semi-constant headspace
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concentration. Shaking was achieved by incubating the microcosms horizontally on a
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rotary shaker (120 rpm). All incubations were performed at 22 °C. Unless stated
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otherwise, at the end of each treatment, slurry samples (~2 g, after thorough mixing)
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were taken for molecular analysis. This was followed by non-sterile surface water
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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
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At the end of the third VC spike under oxic static incubation, 7 mL slurry from replicate
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P25 and P26 microcosms was transferred to 160 mL vials containing 63 mL mineral salts
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medium (MSM)18 for enrichment of aerobic metabolic VC-oxidizing bacteria. The cultures
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were amended with 70 µM VC and sterile oxygen (7 % headspace) as described above
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and incubated static at room temperature. In cultures that depleted VC, 70 µM VC and
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oxygen (if necessary) were re-amended and degradation was followed for two more 6
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consecutive VC spikes. After degradation of the third VC spike, 2 ml samples were taken
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from the suspension for DNA extraction. Subsequently, cultures that showed VC
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degrading activity were transferred to fresh MSM and aerobic VC (70 µM) degradation
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was followed for five transfers. From those highly enriched cultures, 10-1-10-5 dilutions
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were spread onto MSM agar plates and incubated in a desiccator with a 1% (v/v) VC-air
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atmosphere. After two months, individual colonies were aseptically streaked on new MSM
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agar and identically re-incubated in the presence of 1% VC or absence of VC as control.
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Colonies that only showed growth under VC atmosphere were aseptically transferred to
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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
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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)
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DNA was extracted from slurry samples as described.37 qPCR analyses targeting the Dcm
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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,
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vcrA that were below the detection limit of 10 3 copies/g sediment, the detection limit was
181
used as the denominator for normalization.
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etnE MiSeq sequencing
184
A total of 20 samples was selected for etnE amplicon sequencing including time zero
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sediment samples from both locations P25 and P26 as well as samples from the duplicate
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microcosms after selected treatment steps including oxic static, anoxic static-2, anoxic
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shaking, and oxic shaking steps. A 284 bp fragment of the etnE gene that includes the 7
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missense mutations reported to be involved in the transition from ethene- to VC-
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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
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Sequence data
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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
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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
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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
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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
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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
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and interactions and potential synergies with reductive dechlorination in relation to the
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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
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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
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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
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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
