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This study explores biological chlorine cycling in coastal Arctic wet tundra soils. While many previous chlorine-cycling studies have focused on contaminated environments, it is now recognized that chlorine can cycle naturally between inorganic and organic forms in soils. However, these pathways have not previously been described for an Arctic ecosystem. We measured soil organic and inorganic Cl pools, characterized soils and plant tissues with chlorine K-edge X-ray absorption near-edge spectroscopy (Cl-XANES), measured dechlorination rates in laboratory incubations, and analyzed metagenomes and 16S rRNA genes along a chronosequence of revegetated drained lake basins. Concentrations of soil organic chlorinated compounds (Cl=org) were correlated with organic matter content, with a steeper slope in older soils. The concentration and chemical diversity of Cl-org increased with soil development, with Cl-org in younger soils more closely resembling that of vegetation, and older soils having more complex and variable Cl-XANES signatures. Plant Clorg concentrations were higher than previously published values, and can account for the rapid accumulation of Cl-org in soils. The high rates of Cl-org input from plants also implies that soil Cl-org pools turn over many times during soil development. Metagenomic analyses revealed putative genes for synthesis (haloperoxidases, halogenases) and breakdown (reductive dehalogenases, halo-acid dehalogenases) of Cl-org, originating from diverse microbial genomes. Many genome sequences with close similarity to known organohalide respirers (e.g. Dehalococcoides) were identified, and laboratory incubations demonstrated microbial organohalide respiration in vitro. This study provides multiple lines of evidence for a complex and dynamic chlorine cycle in an Arctic tundra ecosystem.
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Biological chlorine cycling in the Arctic Coastal Plain
Jaime E. Zlamal .Theodore K. Raab .Mark Little .Robert A. Edwards .
David A. Lipson
Received: 1 April 2017 / Accepted: 30 July 2017
ÓSpringer International Publishing AG 2017
Abstract This study explores biological chlorine
cycling in coastal Arctic wet tundra soils. While many
previous chlorine-cycling studies have focused on
contaminated environments, it is now recognized that
chlorine can cycle naturally between inorganic and
organic forms in soils. However, these pathways have
not previously been described for an Arctic ecosystem.
We measured soil organic and inorganic Cl pools,
characterized soils and plant tissues with chlorine
K-edge X-ray absorption near-edge spectroscopy (Cl-
XANES), measured dechlorination rates in laboratory
incubations, and analyzed metagenomes and 16S rRNA
genes along a chronosequence of revegetated drained
lake basins. Concentrations of soil organic chlorinated
compounds (Cl
org
) were correlated with organic matter
content, with a steeper slope in older soils. The
concentration and chemical diversity of Cl
org
increased
with soil development, with Cl
org
in younger soils more
closely resembling that of vegetation, and older soils
having more complex and variable Cl-XANES signa-
tures. Plant Cl
org
concentrations were higher than
previously published values, and can account for the
rapid accumulation of Cl
org
in soils. The high rates of
Cl
org
input from plants also implies that soil Cl
org
pools
turn over many times during soil development. Metage-
nomic analyses revealed putative genes for synthesis
(haloperoxidases, halogenases) and breakdown (reduc-
tive dehalogenases, halo-acid dehalogenases) of Cl
org
,
originating from diverse microbial genomes. Many
genome sequences with close similarity to known
organohalide respirers (e.g. Dehalococcoides)were
identified, and laboratory incubations demonstrated
microbial organohalide respiration in vitro. This study
provides multiple lines of evidence for a complex and
dynamic chlorine cycle in an Arctic tundra ecosystem.
Keywords Chlorine Halogen Organohalide
respiration XANES Arctic Dehalococcoides
Introduction
Biogeochemical cycling of naturally occurring chlo-
rinated organic compounds (Cl
org
) has received
increased attention in recent scientific literature
Responsible Editor: Jacques C. Finlay.
Electronic supplementary material The online version of
this article (doi:10.1007/s10533-017-0359-0) contains supple-
mentary material, which is available to authorized users.
J. E. Zlamal M. Little D. A. Lipson (&)
Department of Biology, San Diego State University,
San Diego, CA 92182, USA
e-mail: dlipson@mail.sdsu.edu
T. K. Raab
Carnegie Institution for Science, Stanford,
CA 94305-4101, USA
R. A. Edwards
Department of Computer Science, San Diego State
University, San Diego, CA 92182, USA
123
Biogeochemistry
DOI 10.1007/s10533-017-0359-0
(Biester et al. 2006; Clarke et al. 2009; Leri and
Myneni 2010;O
¨berg 2002; Svensson et al. 2007; Van
den Hoof and Thiry 2012; Weigold et al. 2016).
Chloride (Cl
-
) has generally been considered inert in
ecosystems, and is often used as a conservative tracer
in hydrological studies (Leibundgut et al. 2009).
Microbial chlorine (Cl) metabolism has been studied
mainly in the context of contamination from chlori-
nated compounds used in industry (Asplund and
Grimvall 1991; Hiraishi 2008; Holliger et al. 1997;
Leys et al. 2013; Lohner and Spormann 2013;O
¨berg
2002), and radioactive
36
Cl released following decom-
missioning of nuclear reactors (Bastviken et al. 2013;
Van den Hoof and Thiry 2012). Globally, soil Cl
cycling also has significance for the destructive impact
of methyl halides and other volatile halogenated
organic compounds (VHOC) on the ozone layer
(Keppler et al. 2000; Wetzel et al. 2015). Biogeo-
chemical studies of Cl are rare in general (O
¨berg and
Bastviken 2012). European forests are the best studied
in terms of Cl cycling rates (Bastviken et al. 2009;
Montelius et al. 2016;O
¨berg et al. 2005; Redon et al.
2011; Rohlenova
´et al. 2009; Weigold et al. 2016),
though Cl
org
has also been quantified in grasslands and
farmlands (Redon et al. 2013), and in the pore water of
Chilean peat bogs (Biester et al. 2006). Evidence was
found for biological chlorination of soil organic matter
(SOM) in Canadian peat bogs (Silk et al. 1997). While
several studies have measured VHOC fluxes (Rhew
et al. 2007,2008; Teh et al. 2009), there has been no
previous comprehensive study of internal Cl cycling in
Arctic soils.
Many organisms produce Cl
org
, including fungi,
lichen, bacteria, terrestrial and marine plants and
invertebrates, and even higher animals such as frogs
and mammals (Fielman et al. 1999; Gribble 2003;
Peng et al. 2005). Halogenases and haloperoxidases
catalyze the chlorination of organic compounds
(Bengtson et al. 2013; Niedan et al. 2000; van Pe
´e
and Unversucht 2003). Some halogenated compounds
produced by plants/algae are powerful insecticides,
such as Telfairine produced by the red algae Plo-
camium telfairiae, and many bacterially-produced
antibiotics (such as vancomycin) contain Cl (Gribble
1998). In addition to synthesis of antibiotics, bacteria
may non-specifically halogenate organic compounds
as a form of competitive antagonism or as a defense
against reactive oxygen species (Bengtson et al.
2009,2013). Abiotic processes such as volcanic
eruptions and forest fires produce Cl
org
, and ferrous
iron [Fe(III)] can catalyze the abiotic formation of
organohalogens from halides and organic matter
(Comba et al. 2015; Keppler et al. 2000).
Some bacterial species, such as those in the
Dehalococcoides genus, respire anaerobically using
halogenated organic compounds as the terminal elec-
tron acceptor in organohalide respiration (also referred
to as dehalorespiration, halorespiration, or chlorores-
piration) (Mohn and Tiedje 1992). This process results
in the liberation of Cl
-
from Cl
org
. A variety of
dehalogenase enzymes exist and catalyze slightly
different reactions depending on substrate specificity,
utilizing one of several mechanisms to cleave the bond
between the carbon and halogen atom (Bommer et al.
2014; Kurihara et al. 2000; Rupakula et al. 2013; Tang
and Edwards 2013; Wagner et al. 2013). Organohalide
respiring bacteria often have many different reductive
dehalogenase (RDH) genes (up to 39) in their genome,
probably corresponding to the complex suite of
chlorinated substrates in their environment (Richard-
son 2013; Tang and Edwards 2013).
In wet tundra soils of the Arctic Coastal Plain,
continuous permafrost blocks drainage, and the dom-
inant moss communities on the surface hold soil water
(Brown 1967). The microbial communities in these
waterlogged soils host a diverse range of anaerobic
pathways (Lipson et al. 2013a,2015). Our study site is
located near Barrow, Alaska on the North Slope of the
Arctic Coastal Plain. Much of the landscape is
comprised of thermokarst lakes which drain and
become revegetated slowly over the course of a
roughly 5500 year cycle, with geomorphic stages
defined as young (\50 y.b.p., years before present),
medium (50–300 y.b.p.), old (300–2000 y.b.p.) and
ancient (2000–5500 y.b.p) (Hinkel et al. 2003). As
drained thermokarst lake basins (DTLB) age and
develop, soil carbon accumulates in the surface
organic layer, humic substances increase in complex-
ity, and microtopographic features develop due to the
formation of ice-wedge polygons (Bockheim et al.
2004; Grosse et al. 2013; Hinkel et al. 2005). The
DTLB cycle in the Arctic Coastal Plain provides a
convenient chronosequence to study ecosystem prop-
erties at different stages of soil development (Lipson
et al. 2013b; Sturtevant and Oechel 2013; Zona et al.
2010). The goal of this study was to establish the
internal biological cycling of Cl in soils of the Arctic
Coastal Plain in northern Alaska, and to describe
Biogeochemistry
123
variation in Cl cycling across this chronosequence. We
quantify and chemically characterize soil Cl pools, we
demonstrate the potential for organohalide respiration
in laboratory incubations, and we present 16S rRNA
and metagenomic sequences with high similarity to
the genes and genomes of organisms that participate in
Cl cycling pathways.
Methods
Site description
Soil and soil pore water samples for this study were
collected from DTLB near Barrow, Alaska (centered
around 71.24°N 156.48°W) on the North Slope of the
Arctic Coastal Plain (see Fig. S1, Online Resource 1
for a map of the study area). Sampling occurred during
summers of 2010–2013. The coastal wet tundra near
Barrow is dominated by sedges, grasses and mosses
(Walker et al. 2005), and the soils are characterized by
a seasonally thawed active layer atop deep continuous
permafrost, classified mostly as Aquorthels, Aquitur-
bels, Historthels and Histoturbels (Bockheim et al.
2004; Brown 1967). The depth of the active layer in
these DTLB is around 30–40 cm (Lipson et al. 2013b;
Shiklomanov et al. 2010). As DTLB age, organic
layers develop over silty, organic rich lacustrine
mineral layers (Bockheim et al. 2001). Average
organic layer thickness for the four age classes are 7,
13, 20 and 35 cm for young, medium, old and ancient
DTLB, respectively (Bockheim et al. 2004). Soil pH
declines with age, ranging from an average of 6.12 for
young DTLB to 4.96 for ancient DTLB (Fig. S2,
Online Resource 1). Electrical conductivity also
declines with age, with average values for young and
medium DTLB (407–428 lS/cm) higher than old and
ancient DTLB (180–181 lS/cm, Fig. S2). Other soil
data for representative DTLB along this chronose-
quence have been published previously (Lipson et al.
2013b; Miller et al. 2015).
Soil sampling
Soil samples were collected in June and August of
2011 along 30 m transects in replicate basins of all age
classes. Samples of 3 cm diameter and 20–30 cm
length were collected using coring drill bits and a
handheld power drill. Deeper, 7.5 cm diameter cores
were extracted to a depth of 40 cm using a Snow, Ice
and Permafrost Research Establishment (SIPRE) corer
(with niobium-steel drill bit) and gas-powered engine.
Samples from July 2013 were collected from Y1, Ms,
O1, and A0 basins (Fig. S1, Online Resource 1, and as
described in Sturtevant and Oechel (2013)) using a
long serrated knife. The entire thawed soil profile was
collected up to the depth of the frozen layer (approx-
imately 20 cm). Samples were immediately returned
to the lab and frozen at -40 °C. Samples were shipped
frozen overnight to labs in California by commercial
courier.
Soil solution Cl
-
measurements
Soil pore water samples were collected from a depth of
0–10 cm using installed soil water suction
microlysimeters (Rhizon, Eijkelkamp) from late June
through October 2010 (Lipson et al. 2013b). Seven to
ten spatial replicate samples were collected from a
representative basin of each age class. Cl
-
content was
measured using a colorimetric microplate assay
(Merchant 2009). Plates were read by a Spectra
MAX 190 (Molecular Devices Corp.) at 480 nm and
analyzed using SOFTmax PRO 4.0 software (Life
Sciences Edition by Molecular Devices Corp).
Quantification of Cl pools
Soil samples were analyzed for total halides (TX)
using pyrohydrolysis and Cl
-
titration. The cores
collected in June 2011 were used to study spatial
variability among and within basins (three spatial
replicates from three distinct basins for each of four
age classes, 36 samples total). The SIPRE cores were
used to analyze patterns by depth, using a single deep
soil core from one basin of each age class (four depths
9four classes =16 samples). Depth profiles were
created from SIPRE cores using the following approx-
imate depth increments: 0–5, 10–15, 20–25, and
30–35 cm. Soils were prepared by drying overnight
in a 65 °C drying oven and homogenizing using a
clean mortar and pestle. Plant samples for total organic
halide (TOX) analysis included ten replicates of the
dominant sedge, Carex aquatilis, harvested from
August 12 to September 1, 2007 and duplicate
Sphagnum moss samples harvested from June 11 to
July 18, 2006 from the BE experimental site (Fig. S1,
Online Resource 1). Sphagnum species at this site
Biogeochemistry
123
include S. arcticum,S. tescorum,S. obtusum and S.
orientale (Zona et al. 2011). Between 10 mg and
60 mg of homogenized samples were weighed and
combined with 100–150 mg tungsten powder (100
mesh, Santa Cruz Biotechnology) as a combustion
accelerant before being placed into a clean, new,
ceramic sample boat (COSA Xentaur; Yaphank, NY)
and introduced by the Automatic Boat Controller to a
Mitsubishi Chemical TOX-100 Cl analyzer set up for
TOX analysis by pyrohydrolysis (Asplund et al. 1994)
(see also Fig. S3, Online Resource 1). The gas
(Matheson Inc.) profile was argon (carrier) and
purified oxygen (for combustion). TOX-100 operating
software (ver. 5.1.0.0) was used to analyze the
resulting Cl
-
containing hydrolysate by coulometry.
The method detection limit by coulometry for this
equipment is \100 ppb Cl.
The procedure for TOX was based on Asplund et al.
(1994), except, because our access to the TOX-100
instrument was limited, extractable inorganic Cl (Cl
in
)
was measured using a microplate method and Cl
org
was
obtained by subtraction, rather than the original
approach of directly measuring Cl
org
on leached
subsamples by TOX, and obtaining Cl
in
by subtraction.
Cl
-
measurements were made on subsamples of the
same homogenized soil samples used for TX analyses.
Each sample was shaken for two nights with a 1:10 or
1:20 ratio of dry soil to potassium nitrate solution
acidified with nitric acid (0.2 M KNO
3
, 0.02 M HNO
3
)
to extract Cl
-
. These supernatants were analyzed using
the colorimetric chloride assay described above (Mer-
chant 2009). The Cl
-
content of the extraction solution
(‘‘extraction blank’’) was measured to correct for
inadvertent addition of Cl
-
, and none was detected.
To increase sensitivity of the assay, the ratio of sample
to working reagent was modified, as the original
method was optimized to be linear over a much higher
concentration of chloride (5 mM). Instead, 100 lLof
sample was combined with 50 lL of working reagent,
considerably lowering the background.
Soil Cl concentrations (per unit mass) were con-
verted to a per meter square basis using bulk density
calculated from SOM content using the relationship
derived for these soils in Lipson (2013b). Soil Cl
content measured in the upper *25 cm was extrap-
olated to a nominal active layer depth of 30 cm. To
calculate Cl
org
accumulation in the organic layer along
the chronosequence we used published values of
organic layer C (Bockheim et al. 2004), the
relationship between Cl
org
and SOM derived in the
present study, and a C content of 44% in SOM for
these soils (Brown 1967). Changes in soil Cl pools and
concentrations with soil age were tested using regres-
sion analysis with basin age coded non-parametrically
(young =1, medium =2, old =3 and ancient =4),
as the age categories are non-linear and the exact ages
of the basins used in this study were not known. To
estimate the annual input of Cl
org
from plant growth,
we used published data to estimate net primary
productivity (NPP) in DTLB of each age class. Gross
primary productivity (GPP) was reported for young,
medium and old DTLB’s (Zona et al. 2010). To
convert GPP to NPP, a factor of 0.636 was used based
on a model comparison study for the region (Fisher
et al. 2014). NPP was converted from C units to dry
plant biomass assuming a plant C content of 40%.
These values were multiplied by plant Cl
org
and Cl
in
concentrations from the present study. Relative con-
tributions of vascular plants and mosses were derived
from Zona (2011). Cl
-
inputs in precipitation were
estimated from rain and snow melt data from Liljedahl
(2011), and published Cl
-
content of snow (Jacobi
et al. 2012) and rain (Kalff 1968).
X-ray absorption near-edge spectroscopy
(XANES)
K-edge Cl XANES was performed at energies of
2800–2860 eV to elucidate chemical forms of Cl. The
symmetry of some Cl
-
salts is evident in distinct pre-
edge features, and the position of the pre-edge peak can
be related to the degree of covalency in a metal-Cl
bond. XANES spectra are extremely sensitive to the
immediate neighborhood of Cl in terms of oxidation
state, local symmetry of the absorber, and bond lengths
(Leri et al. 2006,2007). XANES spectra were collected
at Beamline 9-BM-C at the Advanced Photon Source
(Argonne National Laboratory; Lemont, IL) in partial
fluorescence mode in an experimental arrangement
essentially as described in (Bolin 2010) and consisted of
a Si(111)-monochromator, with focusing achieved
using a Rhodium-coated toroidal mirror. Harmonics
were rejected through a flat, Rhodium-coated mirror;
this provided a maximum energy resolution of
0.1–0.2 eV at 2.8 keV. Qualitative exploration of
chlorinated compounds was achieved by linear combi-
nation fitting of normalized soil/plant spectra to a
standard library collected under the identical beamline
Biogeochemistry
123
conditions as the soil spectra (Manceau et al. 2012).
Standards dispersed in boron nitride mulls (BN
3
to
minimize Cl over-absorption) served as Cl-peak energy
standards by which we compared whole soils. Vulcan
Carbon XC72 (Cabot Corporation) was used for
immobilizing organic solvents.
ATHENA software was used to analyze and
compare the spectra (Ravel and Newville 2005). All
sample spectra were normalized after pre-edge and
post-edge corrections. The relative contributions of
known Cl-containing compounds to each spectrum
was analyzed using linear combination fitting (LCF)
(Manceau et al. 2012). Standards were chosen to span
the canonical oxidation states of Cl, and those used to
fit spectra in this study included NaCl, KCl, CaCl
2
,
monochlorodimedone, sucralose, polyvinylchloride,
trichloroethylene, chrome azurol-S, and chloroacetic
acid. LCF analysis produced weights attributed to each
standard, and these weights were used in a Principal
Component Analysis (PCA) to qualitatively compare
the overall similarity of each sample.
Cl XANES was performed on subsamples from the
soil monoliths collected in July 2013. Soils were cut
into horizons with a band saw and dried fully in a
65 °C drying oven. Vegetation samples included the
grass, Arctophila fulva (from the ancient basin, A0),
the sedge, Carex aquatilis (from the medium basin,
Ms), and Sphagnum moss (from the medium basin,
BE). Humic acid extracts from a young and an ancient
basin were prepared as described previously (Lipson
et al. 2013b). Briefly, humic substances from 5 to 10 g
of wet, frozen soil were extracted overnight in N
2
-
bubbled 0.1 M sodium hydroxide (NaOH)/0.1 M
sodium pyrophosphate (Na
4
P
2
O
7
), precipitated by
acidification with hydrochloric acid (HCl) to pH 1,
rinsed with 0.001 M HCl, and finally redissolved in
N
2
-bubbled 50 mM sodium bicarbonate (NaHCO
3
).
To prepare these extracts for XANES, they were
precipitated in 0.1 M sulfuric acid (H
2
SO
4
) and rinsed
with 0.1 lMH
2
SO
4
before being dried at 65 °C. Dried
samples were ground to a fine paste with a clean,
ethanol-rinsed mortar and pestle before mounting onto
carbon tape. Polyethylene glycol was used as a binder
for some soil samples.
Organohalide respiration in laboratory incubations
To measure potential rates of organohalide respiration,
soil slurries were incubated in the laboratory under a
variety of conditions. About 1 g of soil from monoliths
collected from a medium aged basin in July 2013 was
added to 25 mL of sterile, oxygen free, 10 mM pH 5.5
sodium acetate buffer containing 900 lM tetra-
chloroethylene (PCE), 1.8 mM dichloroethylene
(DCE), and 0.1 mg/L thiamine in sterile 50 mL
Wheaton crimp top glass vials fitted with butyl rubber
septa. Septa were secured with metal clamps, and the
headspace was flushed with N
2
gas. Treatments for
this experiment included a matrix of the following:
with and without vitamin B
12
(cobalamin, 0.5 mg/L),
with and without carbon dioxide gas (sufficient to
replace N
2
headspace), and with and without hydrogen
gas as an electron donor (10 cc/vial). The five different
non-sterilized treatments were: (1) ?B
12
,?H
2
,?CO
2
;
(2) ?H
2
,?CO
2
; (3) ?B
12
,?H
2
; (4) ?B
12
,?CO
2
; (5)
?CO
2
. A subset of vials was autoclaved as a control
for abiotic Cl liberation. This treatment received
vitamin B
12
, hydrogen and carbon dioxide gases to
allow direct comparison with the most favorable
conditions for reductive dechlorination. Vials were
incubated at 10 °C and liquid samples were extracted
periodically with a syringe, briefly centrifuged to
remove suspended soil particles, and assayed for Cl
-
as described above. Concentrations were normalized
to lmole Cl
-
per gram of wet soil.
Sequencing and bioinformatics
Eight metagenomes were created using two depths
from each of four age classes; each metagenome
library included combined DNA from three spatial
replicate samples taken from different locations along
a 30 m transect to make each metagenome more
representative of spatial variability. Shallow (5–6 cm
depth) and deep (15–16 cm depth) soils were analyzed
from the following basins: Y1, Ms, O1, and A0.
Samples were thawed and processed (*1 g) using
MO BIO PowerSoil
Ò
DNA isolation kit (MO BIO
Cat# 12888-100). DNA was quantified using Quant-iT
pico green dsDNA assay kit (Life Technologies, Cat#
P11496). Environmental DNA samples (500 ng) were
sheared using a Covaris focused-ultrasonicator M220
with a target fragment size of 500 bp. Shotgun
libraries were prepared using Roche rapid library
Lib-L, Multiple-Prep (MV) preparation methods and
multiplex identifier (MID) adaptors. Libraries were
sequenced on a Roche 454 Life Sciences GS Junior
platform at San Diego State University.
Biogeochemistry
123
MID’s, reads with mean quality scores less than 20,
reads less than 60 bp, duplicates, and reads with more
than 1% ambiguous bases were removed. Read ends
with quality scores less than 20 were trimmed from left
and right using PRINSEQ v0.20.4 (Schmieder and
Edwards 2011). Processed sequencing reads were
uploaded to MG-RAST (Meyer et al. 2008) and are
publicly available (Project ID 7998, MG-RAST ID
numbers 4554152.3-4554159.3).
We re-analyzed our previously published metagen-
omes from a medium aged basin as detailed by Lipson
et al. (2013a). These four metagenomes are from four
depths (0–10, 10–20, 20–30, and 30–40 cm); each
metagenome explored pooled soil DNA from four
spatially replicated soil cores (GenBank SRA acces-
sion number SRP020650). The four previously pub-
lished metagenomes had larger coverage than the eight
described in the current study; however, all twelve
were generated using the same sequencing platform
and analyzed using MG-RAST with hit comparisons
to SEED and GenBank databases. To search more
sensitively for reductive dehalogenase (RDH) genes,
we used a Hidden Markov Model (HMM) analysis
using HMMER v3 (Eddy 2011). The RDH gene
family is well-curated (Hug et al. 2013), and has a
defined protein family (TIGR02486). We searched all
open reading frames in the metagenomes using
hmmer3. Resulting sequences were manually
inspected using tblastn searches to exclude probable
false matches (some genes identified in the HMM
search appeared to be epoxyqueuosine reductases, a
related gene family (Payne et al. 2015). We also used a
local BLAST search to identify cmu genes using
BioEdit (because no authentic matches were found
using MG-RAST). Predicted proteins from our
metagenomes were downloaded from MG-RAST
and used to construct a local protein database.
Published cmuABC genes from Methylobacterium
extorquens were queried against this database and
the top hits were then investigated by searching
against the public database.
Pyrosequencing was performed on 16S rRNA gene
amplicons from old and ancient basin soils collected in
June 2011 (Lipson et al. 2015). Four depths were
studied from high/dry and low/wet topographical
features (rims and centers of low-centered ice wedge
polygons). Sample processing, sequencing, and core
amplicon data analysis were performed by the Earth
Microbiome Project (www.earthmicrobiome.org), and
all amplicon and meta-data made public through the
data portal (www.microbio.me/emp) (Gilbert et al.
2010). EMP protocols are available at http://www.
earthmicrobiome.org/emp-standard-protocols/. All
amplicon and metadata is available through the
European Nucleotide Archive (ENA) of the European
Bioinformatics Institute (EBI) (www.ebi.ac.uk/ena,
study ID: ERP010098 and PRJEB9043).
Results
Seasonal changes in soil pore water Cl
-
Soil pore water Cl
-
concentrations were measured in a
single representative DTLB of each age class over the
summer of 2010 (Y0, M0, O0 and A0). Cl
-
concen-
trations in the medium aged basin were higher than in
the other three basins (Fig. 1). Linear regression
analysis shows the medium and ancient basin Cl
-
concentrations increased significantly over the season
(Medium: slope =14.7 lM/day, R
2
=0.168,
P=0.008; Ancient: slope =5.2 lM/day,
R
2
=0.181, P =0.006), while Cl
-
in the young and
old basins did not (Young: slope =-0.67 lM/day,
R
2
=0.003, P =0.75; Old: slope =-0.47 lM/day,
R
2
=0.002, P =0.79). The high slope in the medium
basin was driven by the initial increase early in the
season, after which the concentrations remained
relatively constant.
Quantification of soil and plant Cl pools
The concentrations of total Cl, Cl
org
and Cl
in
were
measured in soils of all age classes (Fig. 2). This
analysis included three replicates within each basin,
Fig. 1 Soil pore water chloride (Cl
-
) concentrations over the
2010 growing season
Biogeochemistry
123
three basins per age class, four age classes, 36 samples
total. Two samples from one of the medium-aged
basins (Ms) had extremely high Cl concentrations and
were identified as statistical outliers by their extreme
Cook’s D values (Fig. 2a, b, c). Omitting these two
outliers, there were significant increases across the age
gradient in total soil Cl (Fig. 2a, P =0.007), Cl
org
(Fig. 2b, P =0.001), but not Cl
in
(Fig. 2c,
P=0.358). The ratio of Cl
org
to total Cl also increased
across the age gradient (Fig. 2d, P =0.029, the two
Ms samples were not outliers in this analysis). The
increase of Cl
org
with soil development is shown
clearly in the relationship between SOM and Cl
org
(Fig. 2e; this analysis includes the data from Fig. 2a–
d, as well as a depth profile through the entire active
layer from one basin of each age class). An analysis of
covariance (ANCOVA) was used to test whether the
relationship between Cl
org
and SOM varies by basin
age. Cl
org
increased with SOM content (P \0.001),
but Cl
org
in old and ancient basins increased more
steeply with SOM than young and medium basin soils
(Fig. 2e, Basin 9SOM interaction, P =0.013). The
choice of combining the youngest two basins and
comparing them to the two oldest basins was justified
by the corrected Akaike Information criterion (AIC
C
),
which compares models by balancing predictive
power against complexity. In the regression with
separate slopes for all four basins the AIC
C
was
580.80. Collapsing the basins into two categories lead
to a significant improvement in AIC
C
(572.48, a
decrease of 2 or more is commonly considered
significant, Burnham and Anderson 2003). This anal-
ysis indicates higher Cl contents in soil organic matter
of old and ancient basins than young and medium aged
basins.
Total soil Cl and Cl
org
tended to decline with depth
(Fig. S4A–B, Online Resource 1), though these
patterns were statistically weak (P =0.037 and
Fig. 2 Analysis of soil Cl
concentrations (lgg
-1
dry
mass) by basin age: aTotal
Cl, bCl
org
,cCl
in
,dCl
org
as
percent of total Cl. eThe
relationship between Cl
org
and soil organic matter by
basin age. Regression lines
are shown for young and
medium (YM,
y=3.75X ?13.0,
r=0.851) versus old and
ancient (OA,
y=6.45X -42.1,
r=0.818) soils
Biogeochemistry
123
0.075, respectively). Some of this variability was due
to the smaller change with depth in the young basin
profile. Cl
org
as a percent of total Cl showed a non-
significant (P =0.166), positive trend with soil depth
(Fig. S4C, Online Resource 1).
To place our measured soil Cl concentrations in a
landscape context, we converted the data in Fig. 2to
an area basis (Fig. 3). Cl
org
in the active layer
increases with basin age (Fig. 3a, P =0.014). Much
of this is accounted for by an increase in Cl
org
associated with the organic layer (calculated from
published soil C data and the Cl
org
vs. SOM relation-
ship in Fig. 2e) as the organic layer develops over time
(Bockheim et al. 2004). To estimate the potential
contribution of plant Cl
org
to the soil pool we
multiplied our measured concentrations for plant leaf
tissue from a medium-aged DTLB (Table 1)by
published values of plant productivity and calculated
the time it would take for this input to account for the
observed increase in the profile (or in the case of young
DTLB’s, the appearance of Cl
org
in the organic layer).
These estimates are within or below the estimated ages
of each age category, and so plants could account for
the initial accumulation of soil Cl
org
. These rates imply
that the growing soil Cl
org
pool turns over many times
during development (*9–25 times for an ancient
basin with respect to plant Cl
org
inputs, and even more
if other atmospheric and microbial inputs are consid-
ered). It is also clear from Fig. 3a that Cl
org
in pre-
existing lacustrine-derived organic matter makes up a
substantial fraction of the total pool. In contrast to
Cl
org
,Cl
-
does not accumulate in soil profiles over
time, and turns over on the order of 10 years with
respect to Cl
-
in precipitation and runoff (Fig. 3b).
Much of the inorganic Cl that is deposited by
precipitation passes through plants before release as
Cl
-
into the soil. Based on our measurements of plant
total Cl concentrations (Table 1) and previously
published productivity data, 63% of the Cl
-
in
precipitation could be absorbed by growing vegetation
(0.99 g Cl m
-2
year
-1
), with 0.84 g Cl m
-2
year
-1
of
this remaining in inorganic form (Fig. 3b).
Cl XANES
Cl-XANES was performed to reveal chemical
structure information of chlorinated compounds in
bulk soils, soil humic acids, and plant tissue. The
energy of the edge (or ‘‘white line’’) feature of
XANES spectra reflects the oxidation state of the Cl
in the sample, and can be used to determine
whether the compound signature is more organic or
inorganic (Leri et al. 2006,2007; Leri and Myneni
2010). The edge occurs at lower energies for
Fig. 3 Input and accumulation of Cl
org
and Cl
-
in soils. aCl
org
measured in the active layer of basins along the chronosequence
(maximum age for each category shown), Cl
org
content of the
organic layer, and the number of years it would take for the Cl
org
in the soil profile to accumulate given only Cl
org
inputs from
plants. bCl
-
measured in the active layer across the
chronosequence (and the average for all basins), Cl
-
associated
with annual plant production, inputs of Cl
-
from precipitation,
and Cl
-
lost in runoff, assuming steady state conditions
Table 1 Total (Cl
tot
), organic (Cl
org
) and inorganic (Cl
in
)
chlorine concentrations in plant biomass (mg per kg dry mass,
standard errors shown in parentheses)
Plant type Cl
tot
Cl
org
Cl
in
Sedge 4420 (324) 243 (17) 4241 (407)
Moss 541 (3) 256 (74) 285 (72)
Sedges samples were Carex aquatilis, moss samples were
Sphagnum spp.
Biogeochemistry
123
organic chlorinated compounds than inorganic salts
(shown by vertical lines in Fig. 4a). Spectral edges
of the young and medium soils and the plant
samples occurred at higher energy than old and
ancient soils, indicating higher proportions of
inorganic Cl in younger soils and vegetation, and
higher contributions of organic Cl in older soils.
The pre- and post-edge features give information on
the chemical bonding environment of the Cl atoms.
Qualitatively, the spectra of the medium and young
soils were similar to spectra of vegetation, while
old and ancient soils were not (Fig. 4a, b). The
spectra of soils, plants, and soil humic acid extracts
were analyzed with linear combination fitting
(LCF), using a variety of organic and inorganic
Cl standards (examples are shown in Fig. 4c).
While our Cl-XANES libraries are too incomplete
to provide a comprehensive, quantitative description
of the types of Cl-containing molecules in these
samples, LCF with our available standards allows
us to compare the Cl chemistry among our samples.
For each sample, LCF yielded the percent contri-
bution of each standard, and these were reduced to
two dimensions using principle components analysis
(PCA, Fig. 4d). Young and medium soil samples
tended to cluster with vegetation samples (moss,
sedge, and grass), demonstrating the similarity in Cl
signature among these samples. Old and ancient soil
spectra were dispersed on the graph, indicating
diversification in soil Cl signatures as soils develop.
Likewise, the spectra from humic acids (relatively
complex and old compounds) extracted from both
young and ancient aged basins were distinct from
the spectra of both soils and vegetation, indicating
the diversification of Cl chemistry during the
humification process.
Fig. 4 Cl-XANES of soils, plant and standards. aSpectra of
representative soil samples from young (Y), medium (M), old
(O) and ancient (A) basins, and grass tissue, bracketed between
organic (chloroform, CHCl
3
) and inorganic Cl (NaCl) standards.
For reference, the locations of the K-edge are shown for CHCl
3
and NaCl with vertical lines. The spectra are stacked arbitrarily
along the Y axis for purposes of comparison. bSpectra of three
plant samples, a sedge, a grass and a moss. cSpectra of several
organic (polyvinyl chloride (PVC), CHCl
3
, dichlorobenzoic
acid (DCBA), trichloroethylene (TCE)) and inorganic Cl
standards. dPrinciple component analysis of curve-fitting
results for spectra of soils, humic acid and plant tissues, using
all relevant standards
Biogeochemistry
123
Organohalide respiration in laboratory incubations
The potential for organohalide respiration in Arctic
Coastal Plain soils was verified by a laboratory experi-
ment measuring the release of Cl
-
in anaerobic slurries of
autoclave-sterilized and non-autoclaved soils, supplied
with a mix of organic chlorinated compounds (PCE and
DCE) that are dechlorinated by various species (Futagami
et al. 2008). Non-sterilized samples received various
combinations of vitamin B
12
(cobalamin, a cofactor
required by Dehalococcoides), H
2
(an energy source) and
CO
2
(required for autotrophic growth). However, no
differences were observed among non-sterilized treat-
ments, and so these are combined in Fig. 4.Innon-
sterilized treatments, Cl
-
increased linearly over the
course of the incubation (slope =0.0012 lmolClg
-1
soil h
-1
,R
2
=0.464), signifying cleavage of carbon-Cl
bonds via organohalide respiration (Fig. 5). Conversely,
autoclaving the soils appeared to liberate Cl
-
initially,
after which there was no significant increase in chloride
(the first measurement occurred prior to autoclaving the
vials; all other measurements were taken after this step).
A Mann–Whitney U test, performed on the slopes of
individual flasks from this experiment, confirmed that the
slopes were significantly different between autoclaved
and non-autoclaved treatments (P =0.002).
Metagenomics and 16S rRNA gene analysis
We used shotgun metagenomes and surveys of 16S
rRNA genes from our sites to assess the genetic
potential for Cl cycling. One of the two metagenomics
data sets and the 16S rRNA data set were previously
published (Lipson et al. 2013a,2015), and the reader is
referred to these publications for a more thorough
description of the microbial communities in the Arctic
Coastal Plain near Barrow, AK. The previously
published metagenome represented a single, med-
ium-aged basin, while the 16S rRNA study included
only old and ancient basins, and so additional
metagenomes were constructed from basins of all
age categories (Table 2). A general taxonomic
description of these new metagenomes is given in
Table S1 (Online Resource 1). Metagenomic data
revealed the presence of Cl cycling genes in different
depths of Arctic soils of all age classes (Table 2).
Matches to genes annotated as haloperoxidases, (pri-
marily non-heme, vanadium-dependent chloroperox-
idases) were found in all but one soil surveyed (a
relatively small metagenome with only 6407
sequences), and included matches to a diverse range
of microbes, including some strict anaerobes
(Table S2, Online Resource 1). Haloperoxidases
catalyze the oxidation of halides by hydrogen peroxide
and are involved in the synthesis of a variety of
halogenated compounds (Winter and Moore 2009).
Sequence matches to halogenases, which catalyze the
halogenation of organic compounds, were found in
young and medium aged basins, primarily at the
shallower depths of the soil profile. Haloacid dehalo-
genases, responsible for liberating halogens from
2-halo carboxylic acids (Goldman et al. 1968), were
found throughout the landscape everywhere except the
shallow ancient soil (a small metagenome with only
4802 sequences). Reductive dehalogenase (RDH)
genes, responsible for transferring electrons to Cl
org
in the final step of organohalide respiration (Holliger
et al. 1999), were found in young and medium soils.
Our initial BLAST search revealed only 8 sequence
matches to RDH genes. The HMM search for RDH
genes resulted in the 21 matches shown in Table 2.
These Cl cycling genes originated from an unexpect-
edly diverse set of genera of Bacteria and Archaea
(Tables S2, S3, S4, and S5, Online Resource 1). For
example, the 21 sequence matches to RDH genes
represented ten genera from seven different phyla
(Table S5, Online Resource 1). The metagenomes
from all depths and age categories also included
numerous matches to the genomes of Cl-cycling
bacteria, such as the obligate organohalide respiring
genus Dehalococcoides (Tas¸ et al. 2010), the
Fig. 5 Chloride concentration (lmole g
-1
soil) over time in
autoclaved sterilized and non-sterilized soils provided with
dichloroethylene (DCE) and tetrachloroethylene (PCE). Regres-
sions lines for the two treatments are shown
Biogeochemistry
123
dechlorinating genus Anaeromyxobacter and the per-
chlorate-reducing genus Dechloromonas (Table 2).
The relative abundance of these three Cl-cycling
genera was fairly constant across the basins and
depths, though Chi squared tests revealed some
significant trends: Dechloromonas was slightly over-
represented in the young soil (P \0.001);
Anaeromyxobacter (P \0.001) and Dechloromonas
(P =0.004) were enriched in shallower samples of the
0–40 cm study, while Dehalococcoides (P \0.001)
was more abundant at depth (Table S6, Online
Resource 1). The metagenomes contained genomic
matches to the methyl halide degrading bacterial
species, Methylobacterium chloromethanicum and
Hyphomicrobium dentrificans, though no authentic
cmuABC genes were found (McDonald et al. 2002).
Ten putative matches to cmu genes were initially
identified, but these proved to be corrinoid methyl-
transferase proteins in strict anaerobes, more likely
associated with acetogenesis than methyl halide
degradation.
The 16S rRNA gene survey showed higher relative
abundance of Dehalococcoides in lower topography,
with a peak at the intermediate depth of 15 cm below
the surface (Fig. 6). The proportion of Dehalococ-
coides sequences varied with topography and depth
(P =0.012 and P =0.011, respectively, R
2
=0.485,
ANOVA on log-transformed relative abundance data).
16S rRNA genes of bacterial and archaeal genera
known for their capacity to dechlorinate halogenated
organic compounds occur in these soils (the number of
sequences detected, out of 2027,920 total, is shown in
parentheses): Delftia (9) (Zhang et al. 2010), Desul-
fobacterium (52) (Egli et al. 1987), Desulfomonile (16)
(Louie and Mohn 1999), Desulfovibrio (36) (Sun et al.
2000), Methanobacterium (10,649) (Egli et al. 1987),
and Methanosarcina (2135) (Holliger et al. 1992).
Notably, four genera found throughout these soils are
capable of both dechlorination and dissimilatory iron
reduction, another critical component of Arctic soil
metabolism: Anaeromyxobacter (199) (He and San-
ford 2002), Desulfitobacterium (1) (Nonaka et al.
2006), Desulfuromonas (67) (Lo
¨ffler et al. 2000), and
Table 2 Numbers of sequences matching chlorine cycling genes and organisms in metagenomes
Metagenome HPO H HADH RDH Anaeromyxobacter Dechloromonas Dehalococcoides Total post-QC
sequences
Young 5–6 cm 6 1 7 0 234 154 89 30,320
Young 15–16 cm 7 0 2 1 85 23 36 9250
Medium 5–6 cm 4 1 6 1 210 67 135 31,309
Medium 15–16 cm 3 0 1 0 66 20 26 6886
Old 5–6 cm 6 0 2 0 143 41 42 15,558
Old 15–16 cm 0 0 1 0 64 24 30 6407
Ancient 5–6 cm 7 0 0 0 41 9 27 4802
Ancient 15–16 cm 4 0 2 0 69 32 20 8765
Medium 0–10 cm 49 5 26 6 1528 410 296 128,370
Medium 10–20 cm 59 2 33 1 1310 438 376 159,070
Medium 20–30 cm 33 3 21 9 727 217 301 90,764
Medium 30–40 cm 29 0 20 3 721 210 321 79,096
Total 207 12 121 21 1645 5198 1699 570,597
HPO haloperoxidase, Hhalogenase, HADH haloacid dehalogenase, RDH reductive dehalogenase
Fig. 6 Relative abundance of Dehalococcoides 16S rRNA
sequences by depth along the soil profile in areas of high and low
topography (ice-wedge polygon rims and centers, respectively)
Biogeochemistry
123
Geobacter (33,602) (Sung et al. 2006). Additionally,
59 sequences matching the perchlorate-reducer con-
taining genus Dechloromonas were found throughout
the samples (Achenbach et al. 2001).
Discussion
Substantial concentrations of Cl
org
and the presence of
Cl cycling genes in soils of all basin ages indicate
widespread Cl cycling activity in this ecosystem,
driven by diverse microbial communities. The positive
relationship between Cl
org
and SOM, its increased
slope in older basins, and the increase in Cl
org
along
the chronosequence indicate that Cl
org
accumulates as
soils age and SOM develops. These results are similar
to studies of agricultural and forest soils in Sweden
and France, where Cl
org
(Redon et al. 2011; Svensson
et al. 2007) and chlorination rates (Gustavsson et al.
2012) were positively correlated with SOM. The
Arctic soil Cl
org
values found in the present study
range from 18 to 1016 ppm, with an average of
245 ppm. This average concentration is somewhat
higher than previously published values, for example,
87 ppm in a coniferous forest soil in southeast Sweden
(Svensson et al. 2007), 45–100 ppm for arable,
grassland and forest soils of France (Redon et al.
2013), and 133 ppm for a spruce forest soil in
northwest Denmark (O
¨berg and Grøn 1998). The
relatively high Cl
org
concentrations in these Arctic
peat soils are presumably due to higher levels of SOM,
as well as the natural enrichment of halogens in peat
soils (Biester et al. 2006; Keppler and Biester 2003;
Silk et al. 1997). The average concentrations of Cl
org
in plant tissues found in the current study (243 ppm for
sedge tissue, 256 for moss) were generally higher than
previously published Cl
org
values for plant tissues and
litter, which tend to not exceed 100 ppm (O
¨berg 1998;
O
¨berg and Grøn 1998;O
¨berg et al. 1996). Plant Cl
org
concentrations in our study are several fold higher than
reported values for grass (10 ppm) and moss (60 ppm)
in southeast Sweden (Flodin et al. 1997). This might
be explained by the relatively low salinity of the Baltic
sea compared to the Arctic ocean, although the total
plant Cl concentrations in the two studies are similar
(3200 and 700 ppm for grass and moss in Sweden,
4420 and 541 ppm for sedge and moss in this study).
The Cl
org
fraction of the total soil Cl pool (mean of
61.4% in our study) was similar to the average value
for Swedish boreal forest (68.5%) (Svensson et al.
2007), though somewhat lower than the average for
French forest soils (85%) (Redon et al. 2011). The
relative rates of chlorination and dechlorination vary
in space and time, but the presence of these two
opposing processes in soils may tend to balance soil Cl
pools between Cl
org
and Cl
in
. This idea is consistent
with our observation that the soil Cl
org
pool must turn
over many times during soil development, presumably
cycling between organic and inorganic forms due to
microbial activity. There is opportunity for loss of soil
Cl as Cl
-
during snow melt. During this period, early
in the summer, some fraction of the soil Cl
-
pool
might diffuse upward into the overlying water and exit
as runoff. As discussed below, Cl losses through
VHOC fluxes are probably quite small compared to
the soil Cl
org
pool.
The relative rates of Cl
org
accumulation in soils and
production by plants show that plants contribute an
important fraction of the Cl
org
that accumulates in soils
over time. The similarity of XANES spectra of soils
from young and medium-aged basins to those of
vegetation further shows that mosses and graminoids
contribute to the Cl pools in the early developmental
stages of these soils. A development of soil Cl
org
with
time is indicated by the shift in spectra toward a more
organic signature seen as soils age, and by the
increased variability in spectra of older soils and
humic substances (which represent an older, more
complex fraction of the SOM). The TX data support
this interpretation, as the Cl
org
/total Cl fraction
increases with basin age and SOM is enriched in
Cl
org
in old and ancient basins. Furthermore, the
relatively rapid turnover rate of the soil Cl
org
pool with
respect to plant inputs implies that soil Cl pools cycle
between inorganic and organic forms many times
during development of the older basins, allowing
ample opportunity for chemical transformations of
soil Cl
org
. It follows that, while vegetation is a key
input of soil Cl
org
, this pool transforms into more
complex structures with increased functional group
diversity as a result of microbial activity over the
course of soil development, as others have proposed
(Fahimi et al. 2003; Leri and Myneni 2010; Myneni
2002). The Cl cycle we describe for this Arctic peat
soil contrasts with the Cl cycle in Scandinavian
forests, where the concentration of Cl
org
per unit soil
organic matter increases sharply from the O to A
horizon, indicating that Cl
org
is primarily generated in
Biogeochemistry
123
the soil (Hjelm et al. 1995;O
¨berg 1998). This pattern
was not observed in the present study. The relative
contribution of plants and microbes to initial Cl
org
generation may differ between these ecosystems, but
both exhibit rapid cycling between inorganic and
organic forms of Cl in the soil due to microbial
chlorination and dechlorination reactions.
The soil landscape around Barrow generally
becomes less anoxic with development of ice wedge
polygons that create microtopographic relief, resulting
in more high, dry, oxic areas (Lipson et al. 2013b). Our
metagenomic evidence indicates a prevalence of
reductive dehalogenation pathways, which would be
restricted to anoxic microenvironments. Supporting
this idea, genomic sequences from the obligate
organohalide respiring genus Dehalococcoides are
underrepresented near the surface and highest at depth.
Likewise, 16S rRNA genes of this genus are more
abundant in lower, wetter areas than in high, dry areas,
and are generally less abundant near the surface
(Fig. 5). Conversely, the abundance of haloperoxidase
genes indicates that formation of Cl
org
would be
favored in oxic conditions where H
2
O
2
is present,
possibly as an adaptation to oxygen stress (Bengtson
et al. 2013). Therefore, it is possible that an increase in
aerobic conditions as soils age may contribute to the
increased Cl
org
content of SOM in older soils. Young
and medium basin Cl metabolism may be dominated
by reductive dehalogenation, slowing the accumula-
tion of Cl
org
, while synthesis pathways (e.g., haloper-
oxidases, which require peroxide) may dominate in the
more aerated soils of older basins. Alternatively, the
rate of Cl
org
generation may be slightly faster than the
rate of degradation at all stages of soil development;
simply leading to accumulation of Cl
org
in older soils.
Soil development over the thaw lake cycle in coastal
tundra contrasts with longer-term chronosequences
elsewhere in Alaska. For example, moist nonacidic
tundra exists in some recently glaciated sites of the
foothills of the Brooks Range, but older sites are often
taken over by Sphagnum mosses, producing moist
acidic tundra with increased plant productivity,
microbial activity and more reducing conditions
(Hobbie et al. 2002; Walker et al. 1998). Older acidic
soils with more plant production and reducing condi-
tions would probably host a more active Cl cycle than
in the nonacid soils.
In general, microbial chlorination and dechlorina-
tion reactions probably occur simultaneously in most
areas of the landscape, albeit at different depths in the
soil profile. The sharp increase in Cl
-
observed early
in the summer in the medium basin, M0, (Fig. 1) could
have resulted from net dechlorination of Cl
org
.This
increase is not likely due to concentration of salts by
evaporation, as the other basins were subjected to the
same conditions and did not produce similar trends.
These four basins shared the same seasonal patterns in
water table depth (Lipson et al. 2013b). Also, net
primary productivity and evapotranspiration (ET)
peak later in the growing season, from mid-July to
early-August (Zona et al. 2010), so the Cl
-
increase
early in the season is unlikely to result from ET.
Furthermore, if any basin were to show more rapid ET
it would be the young basins that have the highest plant
productivity (Zona et al. 2010). On the other hand, this
medium basin had much higher Cl
-
levels than the
others, and may have had unique hydrology. For
example, M0 is closer to Elson Lagoon than the others
(Fig. S1, Online Resource 1), and could be more
subject to coastal flooding (Brown et al. 2003;
Reimnitz and Maurer 1979). If Cl
-
were concentrated
at a certain depth, then thawing of this layer could also
give rise to the pattern seen in Fig. 1. Similarly, the
outliers in the soil Cl analysis (Fig. 2a–c) both came
from a single medium-aged basin (Ms, Fig. S1, Online
Resource 1) with a relatively low elevation (3 m
compared to mean of 6.5 m for all basins in this study).
Storm surges of greater than 3 m have been reported
for this region (Lynch et al. 2008).
Cl
-
liberation in the laboratory incubation con-
firmed the organohalide respiring capability of these
Arctic soils in vitro. Interestingly, the process of
autoclaving the experimental control soils in the
laboratory incubation resulted in a large release of
chloride (Fig. 5). Assuming bacterial populations
around 10
9
cells/g soil, an estimated cell volume of
1lm
3
, and the reported range of intracellular Cl
-
concentrations for bacteria (typically hundreds of mM
(Fagerbakke et al. 1999)), it is plausible that
the *1lmol Cl
-
/g soil increase after autoclaving
largely resulted from cytoplasmic Cl
-
, though it is
also possible that autoclaving liberated Cl
-
from dead
OM (such as plant cell walls). Cl
-
concentrations in
the incubation experiment were variable over time; the
fluctuations observed in non-sterilized samples may
point to the dynamic nature of multiple, opposing Cl
metabolic pathways occurring during the incubation.
The lack of response to vitamin B
12
,H
2
,orCO
2
Biogeochemistry
123
indicates these factors were not limiting in these
incubations, or that our technique was not sensitive
enough to detect these effects. It is also unknown
whether the Cl
-
liberated during the incubation
originated from naturally occurring Cl
org
or the PCE/
DCE cocktail. Cl tracing experiments using isotopic
labels would allow for more sensitive measurements
of Cl cycling (Bastviken et al. 2009; Montelius et al.
2016).
Previous studies in this Arctic ecosystem showed
that the soil microbial community and its metagen-
ome are dominated by anaerobic respiratory path-
ways (Lipson et al. 2013a,2015). Fe(III) and humic
substances are important electron acceptors for
anaerobic respiration in soils of the Arctic Coastal
Plain (Lipson et al. 2010,2013b), and can compete
with other anaerobic processes such as methanogen-
esis (Miller et al. 2015). Organohalide respiration
occurs simultaneously with Fe(III) reduction in some
environments (Azizian et al. 2010) though inhibitory
effects of Fe(III) on reductive dechlorination have
been reported (Aulenta et al. 2007; Paul and Smold-
ers 2014). Though the redox potential of Cl
org
is
difficult to gauge due to its complex and varied
chemical composition (Gribble 2003; Montelius et al.
2016), the coexistence of organohalide respiration
with Fe(III) reduction in these soils suggests their
reduction potentials may overlap (Shani et al. 2013).
This is consistent with thermodynamic and empirical
evidence that H
2
thresholds are similar for Fe
reduction and organohalide respiration, both orders
of magnitude lower than the threshold for methano-
genesis (Lo
¨ffler et al. 1999; Lovley et al. 1994). This
implies that, like Fe and humic reduction, organoha-
lide respiration has the potential to inhibit methane
production in this ecosystem by reducing H
2
to very
low levels. Furthermore, Fe(III) and humic sub-
stances can react with Cl
-
to create Cl
org
(Fahimi
et al. 2003; Keppler et al. 2000), potentially increas-
ing the opportunity for Cl cycling in these soils. The
Cl, Fe, and C cycles may be highly intertwined in
this ecosystem. Cl inputs from the Arctic Ocean to
this coastal tundra ecosystem are presumably higher
than inland sites (Frontasyeva and Steinnes 2004;
Simpson et al. 2005). However, there are over
400,000 km of permafrost-affected coastline in the
northern hemisphere, representing 34% of the
world’s coasts (Lantuit et al. 2012), and so even if
Cl cycling only plays a major biogeochemical role in
coastal tundra, this would still represent a globally
important phenomenon.
This is the first study to describe internal Cl cycling
in an Arctic ecosystem. However, fluxes of VHOC
have been previously measured in this ecosystem
(Rhew et al. 2007,2008; Teh et al. 2009). Fluxes of
methyl halides and chloroform are very small com-
pared to the overall soil Cl pools and our estimates of
Cl cycling rates. For example, with respect to Cl
org
content from the current study, gross rates of methyl
chloride uptake in this ecosystem [793 nmoles m
-2
-
days
-1
, Teh (2009)] represent a residence time of
about 24,000 years. For comparison, the potential
dechlorination rates found in our laboratory incuba-
tions, or those reported by Montelius et al. (2016),
were both four orders of magnitude higher than the
methyl chloride uptake rate reported by Teh et al.
(2009). The sources and sinks of methyl halides seem
more related to methane cycling microbes than those
that cycle Cl into and out of organic matter: methyl
halide fluxes were correlated with methane fluxes, and
dry sites were larger sinks than wet sites (Rhew et al.
2007; Teh et al. 2009). Thus, methyl halides are
probably degraded aerobically (McDonald et al.
2002), rather than by reductive dechlorination, and
so the landscape patterns for methyl halides are
different from the overall patterns of chlorination
and dechlorination we propose based on soil redox
conditions.
Many studies of organohalide respiration have
focused on model organisms such as Dehalococcoides
or Desulfitobacterium in contaminated sites. Our
results from this Arctic environment with no major
anthropogenic sources of Cl
org
illustrate a naturally
occurring Cl cycle involving a diverse biological
community. While the range of microorganisms
potentially participating in the Cl cycle is vast, the
limited annotation of genomes restricts the ability to
use genetic insight alone to determine the full diversity
of Cl cycling organisms in the environment. Metage-
nomic analysis relies on the annotation of sequenced
genomes, and many gene functions have not been
verified. For example, we found close sequence
matches to genes from strict anaerobes, such as
Methanosarcina acetivorans and Geobacter uraniire-
ducens, that are annotated as chloroperoxidases. While
peroxidases are found in anaerobic prokaryotes (Pas-
sardi et al. 2007), chlorinated products have not (to our
knowledge) been described in anaerobes (van Pe
´e et al.
Biogeochemistry
123
2006). The genes annotated as chloroperoxidases in the
genomes of strict anaerobes might instead be acid
phosphatases or members of the large alpha/beta
hydrolase family (Xu and Wang 2016). On the other
hand, we are doubtlessly missing many other Cl-
cycling genes that have not yet been described and
annotated in sequenced genomes. Despite this uncer-
tainty, observing putative Cl cycling genes in 11 phyla
ranging from Cyanobacteria to Archaea was one of the
more surprising aspects of the present research.
This is one of the most thorough studies to date on
soil Cl cycling, combining chemical, genetic, field-
based, and laboratory-based results; furthermore, it is
the first to demonstrate the internal cycling of Cl in an
Arctic ecosystem. Significant amounts of Cl
org
enter
these soils through the vegetation, and this pool
accumulates, turns over and diversifies due to micro-
bial activity as soils age. Diverse Cl-cycling microbes
are ubiquitous across this landscape. The Cl cycle has
the potential to interact with the biogeochemical
cycling of Fe and C in this ecosystem. Even if such an
active Cl cycle is mainly restricted to coastal tundra,
this study still represents an important and previously
unrecognized aspect of Arctic biogeochemistry, and
provides further evidence that natural Cl cycling in
ecosystems is a widespread phenomenon.
Acknowledgements Dominic Goria, Matt Haggerty and the
SDSU Ecological Metagenomics Class of 2012 were
instrumental in metagenome creation and analysis. Donatella
Zona and Paulo Olivas provided plant samples. We thank Trudy
Bolin and Tianpin Wu at Argonne National Labs for their
training and patience. This research used resources of the
Advanced Photon Source, a U.S. Department of Energy (DOE)
Office of Science User Facility operated for the DOE Office of
Science by Argonne National Laboratory under Contract No.
DE-AC02-06CH11357. XANES spectra were collected at the
X-ray Operations and Research Beamline 9-BM-C at the
Advanced Photon Source, Argonne National Laboratory in
Lemont, Illinois, U.S.A. Competitive access to the APS was
greatly aided by proof-of-concept XANES spectra collected at
the Canadian Light Source by Yongfeng Hu. This work was
supported in part by National Science Foundation Grants
0808604, 1204263 and 1712774.
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Biogeochemistry
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... Interest in applying reductive dechlorination for the bioremediation of chlorinated contaminants has led to the purification and characterization of reductive dehalogenase proteins (reviewed by Jugder et al., 2015;Fincker and Spormann, 2017) and further identification of many more reductive dehalogenase genes (RDases) detected via DNA analysis (e.g., Adrian et al., 2007b;Krajmalnik-Brown et al., 2007;Tang et al., 2013). Putative RDase genes and organohalide-respiring bacteria have been found in uncontaminated environments, including marine sediment (e.g., Futagami et al., 2013;Kawai et al., 2014;Marshall et al., 2017), soil (e.g., Krzmarzick et al., 2012;Weigold et al., 2016), arctic soil (e.g., Zlamal et al., 2017), lake water (e.g., Biderre-Petit et al., 2016), and lake sediment (e.g., Krzmarzick et al., 2013), where they are thought to respire Cl-NOM. Nevertheless, it is unclear whether the amendment of Cl-NOM can enrich for RDases and whether the organisms involved in Cl-NOM dechlorination are more likely to be obligate or facultative organohalide respiring bacteria. ...
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Organohalide-respiring bacteria have been linked to the cycling and possible respiration of chlorinated natural organic matter (Cl-NOM) in uncontaminated soils and sediments. The importance of non-respiratory hydrolytic/oxidative dechlorination processes in the cycling of Cl-NOM in terrestrial soil and sediment, however, is still not understood. This research analyzes the dechlorination potential of terrestrial systems through analysis of the metagenomes of urban lake sediments and cultures enriched with Cl-NOM. Even with the variability in sample type and enrichment conditions, the potential to dechlorinate was universal, with reductive dehalogenase genes and hydrolytic or oxidative dehalogenase genes found in all samples analyzed. The reductive dehalogenase genes detected grouped taxonomically with those from organohalide-respiring bacteria with broad metabolic capabilities, as opposed to those that obligately respire organohalides. Furthermore, reductive dehalogenase genes and two haloacid dehalogenase genes increased in abundance when sediment was enriched with high concentrations of Cl-NOM. Our data suggests that both respiratory and non-respiratory dechlorination processes are important for Cl-NOM cycling, and that non-obligate organohalide-respiring bacteria are most likely involved in these processes.
... Genomics and allied technologies have greatly increased the diversity of putative rdh genes in recent years, and extended their distribution from contaminated environments to deep subsurface (Table 1), Antarctic soils (Zlamal et al. 2017), and even human and animal intestinal tract (Atashgahi et al. 2018b). With the expanding availability of the bacterial genomes and increasing application of deep sequencing in diverse environments, much more diverse and likely novel rdh genes are expected in future. ...
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Attempts for bioremediation of toxic organohalogens resulted in the identification of organohalide-respiring bacteria harbouring reductive dehalogenases (RDases) enzymes. RDases consist of the catalytic subunit (RdhA, encoded by rdhA) that does not have membrane-integral domains, and a small putative membrane anchor (RdhB, encoded by rdhB) that (presumably) locates the A subunit to the outside of the cytoplasmic membrane. Recent genomic studies identified a putative rdh gene in an uncultured deltaproteobacterial genome that was not accompanied by an rdhB gene, but contained transmembrane helixes in N-terminus. Therefore, rather than having a separate membrane anchor protein, this putative RDase is likely a hybrid of RdhA and RdhB, and directly connected to the membrane with transmembrane helixes. However, functionality of the hybrid putative RDase remains unknown. Further analysis showed that the hybrid putative rdh genes are present in the genomes of pure cultures and uncultured members of Bacteriodetes and Deltaproteobacteria, but also in the genomes of the candidate divisions. The encoded hybrid putative RDases have cytoplasmic or exoplasmic C-terminus localization, and cluster phylogenetically separately from the existing RDase groups. With increasing availability of (meta)genomes, more diverse and likely novel rdh genes are expected, but questions regarding their functionality and ecological roles remain open.
... Genomics and allied technologies have greatly increased the diversity of putative rdh genes in recent years, and extended their distribution from contaminated environments to deep subsurface (Table 1), Antarctic soils (Zlamal et al. 2017), and even human and animal intestinal tract (Atashgahi et al. 2018b). With the expanding availability of the bacterial genomes and increasing application of deep sequencing in diverse environments, much more diverse and likely novel rdh genes are expected in future. ...
Article
Full-text available
Attempts for bioremediation of toxic organohalogens resulted in the identification of organohalide-respiring bacteria harbouring reductive dehalogenases (RDases) enzymes. RDases consist of the catalytic subunit (RdhA, encoded by rdhA) that does not have membrane-integral domains, and a small putative membrane anchor (RdhB, encoded by rdhB) that (presumably) locates the A subunit to the outside of the cytoplasmic membrane. Recent genomic studies identified a putative rdh gene in an uncultured deltaproteobacterial genome that was not accompanied by an rdhB gene, but contained transmembrane helixes in N-terminus. Therefore, rather than having a separate membrane anchor protein, this putative RDase is likely a hybrid of RdhA and RdhB, and directly connected to the membrane with transmembrane helixes. However, functionality of the hybrid putative RDase remains unknown. Further analysis showed that the hybrid putative rdh genes are present in the genomes of pure cultures and uncultured members of Bacteriodetes and Deltaproteobacteria, but also in the genomes of the candidate divisions. The encoded hybrid putative RDases have cytoplasmic or exoplasmic C-terminus localization, and cluster phylogenetically separately from the existing RDase groups. With increasing availability of (meta)genomes, more diverse and likely novel rdh genes are expected, but questions regarding their functionality and ecological roles remain open.
... 29,30 These degraded forested wetlands, with large amounts of organic matter from fallen branches and leaves and additional halogen input from seawater intrusion, have a significantly different biogeochemical environment in comparison to the adjacent ecosystems. The interaction of halogens with organic-rich sediments can result in the formation of organohalogens, 31,32 thus making the degraded forested wetland potentially a hotspot for halocarbon emissions. However, only one set (n = 4) of chloroform fluxes from this degraded ecosystem have been reported thus far, and the fluxes were on average higher than the bracketing freshwater wetland and saltmarsh ecosystems, an observation that was also consistent with soil core incubations from these same ecosystems. ...
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Tropical and subtropical-storm surges combined with sea level rise cause saltwater intrusions into low-lying coastal ecosystems along the southeastern coast of the USA, gradually converting freshwater forested wetland into salt marsh. The transition zone between freshwater and saltwater ecosystems becomes a degraded forested wetland, where the combination of high levels of soil organic matter and elevated concentrations of halide ions creates a dynamic biogeochemical environment that may be a potential hotspot for halocarbon formation such as chloroform, methyl chloride and methyl bromide. This study conducted field measurements at a transition zone in coastal South Carolina to quantify halocarbon exchange rates, and laboratory soil incubations to determine the contributions of biotic versus abiotic processes. The degraded forested wetland showed significant chloroform emission rates (146 ± 129 nmol m-2 d-1). The degraded forested wetland remained a net sink for methyl chloride and a negligible source/sink for methyl bromide, unlike the salt marsh which was a significant source for both. The laboratory incubations strongly suggest that methyl halide consumption in soils at the field site was biotic and that production of methyl halides and chloroform was largely abiotic and temperature-dependent, although additional experiments are required to rule out possible biotic production involving heat-resistant microbes. The results suggest that sea level rise and more frequent storm surges derived from global climate change, in the long term, may increase emissions of chloroform from coastal degraded forested wetlands and of methyl halides if salt marshes expand, with potential impacts for stratospheric ozone depletion.
... The ability for bacteria in natural soils to dechlorinate has been shown through the addition and subsequent dechlorination of anthropogenic chemicals (e.g. Zlamal et al. 2017), and in situ dechlorination rates have been estimated through the use of 36 Cl tracers and X-ray absorption near edge structure (XANES) of the Cl-C spectral signatures (Leri and Myneni 2010;Montelius et al. 2016). None of these studies linked specific bacterial groups to the dechlorination activities. ...
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Fungal chloroperoxidases (CPOs) are one class of enzymes that produce natural organochlorides in soils. The microbial degradation of these organochlorides is not well known, though has implications for bioremediation, microbial ecology, and natural chlorine and carbon cycling. In this study, Illumina-based 16S rRNA gene sequencing and real-time quantitative PCR (qPCR) was used to characterize the bacterial community enriched from an amendment of organic matter reacted with CPO under conditions conducive towards chlorination (CPO-OM). In total, 17 bacterial groups were enriched in triplicate microcosms inoculated with creek sediment and amended with CPO-OM. These bacterial groups were not enriched with amendments of non-reacted organic matter extract, with or without oxidative stress induced by H2O2, nor with amendments of organic matter reacted with CPO under non-chlorinating conditions. Of these, only two represented genera with known organohalide respiring bacteria (OHRB)—Dehalogenimonas and Dehalobacter. The genus Acetobacterium was also found to be enriched but the other 14 groups of enriched bacteria do not currently have any close phylogenetically related isolates. This study highlights a gap in the current understanding of the microbiology involved in natural organochloride turnover and suggests that CPO-OM could be used for isolating and culturing strains from novel bacteria genera.
Article
Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 (‘JG233’), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.
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Chlorine (Cl) in the terrestrial environment is of interest from multiple perspectives, including the use of chloride as a tracer for water flow and contaminant transport, organochlorine pollutants, Cl cycling, radioactive waste (radioecology; ³⁶ Cl is of large concern) and plant science (Cl as essential element for living plants). During the past decades, there has been a rapid development towards improved understanding of the terrestrial Cl cycle. There is a ubiquitous and extensive natural chlorination of organic matter in terrestrial ecosystems where naturally formed chlorinated organic compounds (Cl org ) in soil frequently exceed the abundance of chloride. Chloride dominates import and export from terrestrial ecosystems while soil Cl org and biomass Cl can dominate the standing stock Cl. This has important implications for Cl transport, as chloride will enter the Cl pools resulting in prolonged residence times. Clearly, these pools must be considered separately in future monitoring programs addressing Cl cycling. Moreover, there are indications that (1) large amounts of Cl can accumulate in biomass, in some cases representing the main Cl pool; (2) emissions of volatile organic chlorines could be a significant export pathway of Cl and (3) that there is a production of Cl org in tissues of, e.g. plants and animals and that Cl can accumulate as, e.g. chlorinated fatty acids in organisms. Yet, data focusing on ecosystem perspectives and combined spatiotemporal variability regarding various Cl pools are still scarce, and the processes and ecological roles of the extensive biological Cl cycling are still poorly understood.
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Recent work revealed an active biological chlorine cycle in coastal Arctic tundra of northern Alaska. This raised the question whether chlorine cycling was restricted to coastal areas, or if these processes extended to inland tundra. The anaerobic process of organohalide respiration, carried out by specialized bacteria like Dehalococcoides, consumes hydrogen gas and acetate using halogenated organic compounds as terminal electron acceptors, potentially competing with methanogens that produce the greenhouse gas, methane. We measured microbial community composition and soil chemistry along a ~262 km coastal-inland transect to test for the potential of organohalide respiration across the Arctic Coastal Plain, and studied the microbial community associated with Dehalococcoides to explore the ecology of this group and its potential to impact C cycling in the Arctic. Brominated organic compounds declined sharply with distance from the coast, but decrease in organic chlorine pools was more subtle. The relative abundance of Dehalococcoides was similar across the transect, except being lower at the most inland site. Dehalococcoides correlated with other strictly anaerobic genera, plus some facultative ones, that had the genetic potential to provide essential resources (hydrogen, acetate, corrinoids, or organic chlorine). This community included iron reducers, sulfate reducers, syntrophic bacteria, acetogens and methanogens, some of which might also compete with Dehalococcoides for hydrogen and acetate. Throughout the Arctic Coastal Plain, Dehalococcoides is associated with the dominant anaerobes that control fluxes of hydrogen, acetate, methane and carbon dioxide. Depending on seasonal electron acceptor availability, organohalide respiring bacteria could impact carbon cycling in Arctic wet tundra soils. Importance Once considered relevant only in contaminated sites, it is now recognized that biological chlorine cycling is widespread in natural environments. However, linkages between chlorine cycling and other ecosystem processes are not well established. Species in the genus Dehalococcoides are highly specialized, using hydrogen, acetate, vitamin B12-like compounds and organic chlorine produced by the surrounding community. We studied which neighbors might produce these essential resources for Dehalococcoides species. We found that Dehalococcoides are ubiquitous across the Arctic Coastal Plain and are closely associated with a network of microbes that produce or consume hydrogen or acetate, including the most abundant anaerobic bacteria and methanogenic archaea. We also found organic chlorine and microbes that can produce these compounds throughout the study area. Therefore, Dehalococcoides could control the balance between carbon dioxide and methane (a more potent greenhouse gas) when suitable organic chlorine compounds are available to drive hydrogen and acetate uptake.
Chapter
The widespread global distribution of chlorinated hydrocarbons, their high lipophilicity and their recalcitrance have contributed to their importance as environmental toxicants. Their metabolism under oxic and anoxic conditions mediated by prokaryotic and eukaryotic cells is discussed. Various aerobic bacteria are able to use chlorinated hydrocarbons as the sole source of carbon and energy and some anaerobic bacteria can use some of these as an artificial electron acceptor in reductive dechlorination. Liver enzymes are responsible for the formation of hydrophilic metabolites ready for excretion which often lead to highly reactive and potentially toxic intermediates. In contrast, fungi, especially ligninolytic ones, usually only exhibit ‘side activities’ for the transformation of chlorinated hydrocarbons. The capabilities of bacteria had led to the development of various bioremediation processes. Both, successes and failures within these processes are known. Therefore, current research aims at a better understanding of global community interactions. Key Concepts • Chlorinated hydrocarbons have been produced by the chemical industry since decades in large amounts, but they can also be observed as natural compounds, sometimes exceeding the industrially produced amounts. • Even though toxicological properties have pushed the chlorochemistry into the focus of considerable debate and governmental regulatory action, chlorinated hydrocarbons remain essential for certain applications. • The aerobic metabolism of chlorinated hydrocarbons by bacteria has been studied in detail and an immense amount of information is available on pathways, enzymes and genes involved in the mineralisation of chloroaromatics. • The anaerobic degradation of chlorinated hydrocarbons is due to the capability of anaerobic bacteria to use them in anaerobic respiration, which results in dechlorination. • Dehalococcoides organisms are the most versatile reductive dehalogenators as being capable to dehalogenate chlorinated dioxins, biphenyls, benzenes and vinyl chloride, among others. • Microbial activities have been widespread used for bioremediation purposes through natural attenuation, biostimulation and bioaugmentation. • The poor understanding of the functioning of the complex microbial activities in situ made bioremediation efforts quite unreliable. • The rapid development of molecular techniques in recent years allows immense insights into the processes in situ , but also on the overall physiology of biocatalysts.
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Organic and inorganic chlorine compounds are formed by a broad range of natural geochemical, photochemical and biological processes. In addition, chlorine compounds are produced in large quantities for industrial, agricultural and pharmaceutical purposes, which has led to widespread environmental pollution. Abiotic transformations and microbial metabolism of inorganic and organic chlorine compounds combined with human activities constitute the chlorine cycle on Earth. Naturally occurring organochlorines compounds are synthesized and transformed by diverse groups of (micro)organisms in the presence or absence of oxygen. In turn, anthropogenic chlorine contaminants may be degraded under natural or stimulated conditions. Here, we review phylogeny, biochemistry and ecology of microorganisms mediating chlorination and dechlorination processes. In addition, the co-occurrence and potential interdependency of catabolic and anabolic transformations of natural and synthetic chlorine compounds are discussed for selected microorganisms and particular ecosystems.
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In soils halogens (fluorine, chlorine, bromine, iodine) are cycled through the transformation of inorganic halides into organohalogen compounds and vice versa. There is evidence that these reactions are microbially driven but the key enzymes and groups of microorganisms involved are largely unknown. Our aim was to uncover the diversity, abundance and distribution of genes encoding for halogenating and dehalogenating enzymes in a German forest soil by shotgun metagenomic sequencing. Metagenomic libraries of three soil horizons revealed the presence of genera known to be involved in halogenation and dehalogenation processes such as Bradyrhizobium or Pseudomonas. We detected a so far unknown diversity of genes encoding for (de)halogenating enzymes in the soil metagenome including specific and unspecific halogenases as well as metabolic and cometabolic dehalogenases. Genes for non-heme, no-metal chloroperoxidases and haloalkane dehalogenases were the most abundant halogenase and dehalogenase genes, respectively. The high diversity and abundance of (de)halogenating enzymes suggests a strong microbial contribution to natural halogen cycling. This was also confirmed in microcosm experiments in which we quantified the biotic formation of chloroform and bromoform. Knowledge on microorganisms and genes that catalyze (de)halogenation reactions is critical because they are highly relevant to industrial biotechnologies and bioremediation applications.
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Halogenated natural products are widespread in the environment, and the halogen atoms are typically vital to their bioactivities. Thus far, six families of halogenating enzymes have been identified: cofactor-free haloperoxidases (HPO), vanadium-dependent haloperoxidases (V-HPO), heme iron-dependent haloperoxidases (HI-HPO), non-heme iron-dependent halogenases (NI-HG), flavin-dependent halogenases (F-HG), and S-adenosyl-L-methionine (SAM)-dependent halogenases (S-HG). However, these halogenating enzymes with similar biological functions but distinct structures might have evolved independently. Phylogenetic and structural analyses suggest that the HPO, V-HPO, HI-HPO, NI-HG, F-HG, and S-HG enzyme families may have evolutionary relationships to the α/β hydrolases, acid phosphatases, peroxidases, chemotaxis phosphatases, oxidoreductases, and SAM hydroxide adenosyltransferases, respectively. These halogenating enzymes have established sequence homology, structural conservation, and mechanistic features within each family. Understanding the distinct evolutionary history of these halogenating enzymes will provide further insights into the study of their catalytic mechanisms and halogenation specificity.
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Environmental context Natural organohalogens produced in and released from soils are of utmost importance for ozone depletion in the stratosphere. Formation mechanisms of natural organohalogens are reviewed with particular attention to recent advances in biomimetic chemistry as well as in radical-based Fenton chemistry. Iron-catalysed oxidation in biotic and abiotic systems converts organic matter in nature to organohalogens. Abstract Natural and anthropogenic organic matter is continuously transformed by abiotic and biotic processes in the biosphere. These reactions include partial and complete oxidation (mineralisation) or reduction of organic matter, depending on the redox milieu. Products of these transformations are, among others, volatile substances with atmospheric relevance, e.g. CO2, alkanes and organohalogens. Natural organohalogens, produced in and released from soils and salt surfaces, are of utmost importance for stratospheric (e.g. CH3Cl, CH3Br for ozone depletion) and tropospheric (e.g. Br2, BrCl, Cl2, HOCl, HOBr, ClNO2, BrNO2 and BrONO2 for the bromine explosion in polar, marine and continental boundary layers, and I2, CH3I, CH2I2 for reactive iodine chemistry, leading to new particle formation) chemistry, and pose a hazard to terrestrial ecosystems (e.g. halogenated carbonic acids such as trichloroacetic acid). Mechanisms for the formation of volatile hydrocarbons and oxygenated as well as halogenated derivatives are reviewed with particular attention paid to recent advances in the field of mechanistic studies of relevant enzymes and biomimetic chemistry as well as radical-based processes.
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Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5prime-GUN-3prime tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbours a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveal an unexpected diversity in the reductive chemistry catalyzed by these enzymes.
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Methane–climate interactions are reasonably well understood; the biogeochemical controls on net methane fluxes are less so. Within anoxic soils, alternative electron acceptors such as iron and humic substances influence microbial metabolic function, and thus affect the amount of carbon lost as methane (CH4). We present three years of data from wet sedge tundra landscapes near Barrow, Alaska that show an inverse relationship between dissolved iron and CH4 concentrations. We found increasing organic layer thickness related to increases in active layer organic matter content, and decreases in both bulk density and extractable iron. Organic layer depth was also a good proxy for carbon dioxide (CO2) and CH4 dynamics, with increasing organic layer depths relating to lower dissolved iron, higher amounts of dissolved CH4, and lower CO2:CH4 ratios in the upper active layer. Net CH4 fluxes were also significantly suppressed following the experimental addition of iron and humic acids. Iron and humic acid treatment effects were indistinguishable for CH4 net flux; in contrast, post-treatment CH4 fluxes were an average of 0.74-fold the control treatment flux rates. These results suggest that in-situ CH4 production is tied to alternative electron acceptor availability, and that organic layer thickness is a good predictor of biogeochemical controls on CH4 fluxes in wet-sedge Arctic Alaskan tundra.
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Much of the total pool of chlorine (Cl) in soil consists of naturally produced organic chlorine (Clorg). The chlorination of bulk organic matter at substantial rates has been experimentally confirmed in various soil types. The subsequent fates of Clorg are important for ecosystem Cl cycling and residence times. As most previous research into dechlorination in soils has examined either single substances or specific groups of compounds, we lack information about overall bulk dechlorination rates. Here we assessed bulk organic matter chlorination and dechlorination rates in coniferous forest soil based on a radiotracer experiment conducted under various environmental conditions (additional water, labile organic matter, and ammonium nitrate). Experiment results were used to develop a model to estimate specific chlorination (i.e., fraction of Cl(-) transformed to Clorg per time unit) and specific dechlorination (i.e., fraction of Clorg transformed to Cl(-) per time unit) rates. The results indicate that chlorination and dechlorination occurred simultaneously under all tested environmental conditions. Specific chlorination rates ranged from 0.0005 to 0.01 d(-1) and were hampered by nitrogen fertilization but were otherwise similar among the treatments. Specific dechlorination rates were 0.01-0.03d(-1) and were similar among all treatments. This study finds that soil Clorg levels result from a dynamic equilibrium between the chlorination and rapid dechlorination of some Clorg compounds, while another Clorg pool is dechlorinated more slowly. Altogether, this study demonstrates a highly active Cl cycling in soils.
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More than 1500 different halogenated chemicals are produced and discharged into our biosphere by plants, marine organisms, insects, bacteria, fungi, and other natural processes. In a few cases, the quantities of these naturally occurring halogenated compounds far exceed the amounts of the same chemicals from anthropogenic sources. Furthermore, the evidence is overwhelming that natural enzymatic, thermal, and other processes are constantly occurring in the oceans, in the atmosphere, and in the soil that lead to the formation of chlorinated and brominated phenols and many other halogenated chemicals, including dioxins, that previously were thought only to result from the actions of man. Moreover, it is clear that these natural processes have been producing halogenated compounds and have been an important component of our ecosystem for eons.