Stereoselective Microbial Dehalorespiration with Vicinal Dichlorinated Alkanes
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2003, p. 5643–5647 Vol. 69, No. 9
0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.9.5643–5647.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Stereoselective Microbial Dehalorespiration with Vicinal
Dichlorinated Alkanes
Stefaan De Wildeman,1 Gabriele Diekert,2 Herman Van Langenhove,3
and Willy Verstraete1*
Laboratory for Microbial Ecology and Technology1 and Laboratory of Organic Chemistry,3
Ghent University, B-9000 Ghent, Belgium, and Institute for Microbiology,
Friedrich Schiller University Jena, D-07743 Jena, Germany2
Received 10 March 2003/Accepted 1 July 2003
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The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C2 groundwater
pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist.
Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reduc-
tive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and
metabolically convert them into completely dechlorinated end products in defined growth media have been
reported. Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally
defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible
vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identi-
fied as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chloro-
substrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-
conserving anti mechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does
not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This
unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation
purposes and fine-chemical synthesis.
1,2-Dichloroethane (1,2-DCA) is used as an intermediate of can partially convert 1,2-DCA into ethene in an energy-con-
industrial polyvinyl chloride production. Due to leakage and serving manner (16, 17), indicating a typically fast dehalore-
improper disposal, this compound has become the most abun- spiratory process (9). However, this overall dichloroelimina-
dant chlorinated C2 groundwater pollutant on earth (the top tion reaction concomitantly produces up to 1% carcinogenic
five chlorinated and brominated C2 solvents based on average vinyl chloride (VC) (15), while strain 195 depends on unknown
annual underground releases from 1988 to in the United States bacterial extracts. Since VC is also dechlorinated by strain 195
are 1,2-dichloroethane [136.6 tons/year], tetrachloroethene under certain conditions in a cometabolic process, the possi-
[9.2 tons/year], 1,2-dibromoethane [0.6 tons/year], 1,1,1-tri- bility that this compound is an intermediate of the 1,2-DCA
chloroethane [0.5 tons/year], and trichloroethene [0.3 tons/ conversion cannot be excluded (18). In mixed bacterial cul-
year] [see “toxics release inventory” at U.S. Environmental tures, the dichloroelimination of the analogous pollutant 1,2-
Protection Agency website http://www.epa.gov/triexplorer dichloropropane (1,2-D) was linked to thus-far-uncultivated
/chemical.htm]). Due to its high water solubility (8 g/liter), its and unknown Dehalococcoides and Dehalobacter species (13,
environmental half-life of 50 years in anoxic aquifers (28), and 14, 22, 23). So far, there have been no reports of characterized
its probably carcinogenic effects (21), this pollutant poses a bacteria in defined media that completely dechlorinate 1,2-
dispersing and long-lasting danger for humans and wildlife. DCA or 1,2-D in a dehalorespiratory process without produc-
Mostly, pump-and-treat technologies are too costly and time- ing any chlorinated coproducts. We describe the isolation and
intensive to remediate expanded contamination plumes, while characterization of a bacterium that fulfills these criteria for
oxidative in situ (bio)conversion is not compatible with the 1,2-DCA, 1,2-D, and other chloroalkanes. The bacterium,
prevailing groundwater conditions. There is no reductive in which has possible environmental applications, is the first to
situ (bio)remediation process for 1,2-DCA, as there is for link dehalorespiration to stereochemistry.
many other chlorinated pollutants (1, 11, 16, 25), making its
removal problematic.
MATERIALS AND METHODS
1,2-DCA can undergo several cometabolic microbial and
Bacterial inoculum and growth media. The starting bacterial inoculum was
abiotic dechlorination reactions in anoxic habitats (Fig. 1), but
obtained from the soil matrix of an anoxic water-saturated layer (1 m in depth)
these conversions are slow and incomplete (8, 27, 29). Only that had been exclusively polluted with 1,2-DCA (50 mg/kg) for 30 years (soil
one isolated anaerobe, Dehalococcoides ethenogenes strain 195, under an industrial storage tank containing 1,2-DCA). No other chlorinated
substrates were present. The inoculum (1%, wt/vol) was transferred to sterilized
anaerobic media containing 40 mM electron donor, 400 M 1,2-DCA (sole
electron acceptor since CO2 could not be reduced by strain DCA1), 0.02%
* Corresponding author. Mailing address: Faculty of Agriculture (wt/vol) yeast extract (YE), vitamins [300 nM vitamin B1, 800 nM vitamin B3, 40
and Applied Biological Sciences, Ghent University, Coupure Links nM vitamin B7, 40 nM vitamin B12, 300 nM vitamin H1, 100 nM Ca-D(⫹)-
653, B-9000 Ghent, Belgium. Phone: 32 9 2645976. Fax: 32 9 2646248. pantothenate, 600 nM pyridoxamine 䡠 2HCl], trace elements (20 nM
E-mail: willy.verstraete@ugent.be. N2SeO3䡠5H2O, 10 nM Na2WO4䡠2H2O, 7.2 M FeSO4䡠7H2O, 1 M ZnCl2, 0.2
56435644 DE WILDEMAN ET AL. APPL. ENVIRON. MICROBIOL.
coccus and a curved rod. The coccus could be isolated from
this bibacterial culture and was identified as an Enterococcus
casseliflavus strain. This organism showed no dechlorination
capacity. Hence, it was assumed that the curved rod was the
dechlorinating organism.
A liquid medium dilution series of the bibacterial culture
suggested a synergistic interaction between E. casseliflavus and
the curved rod. Growth and dechlorination were observed only
FIG. 1. Possible dechlorination reactions of 1,2-DCA in anoxic in dilution cultures containing both types of bacteria. Indeed,
habitats: dichloroelimination (A), reductive hydrogenolysis (B), and media that only contained the curved rod, which was visible
dehydrochlorination (C). under the microscope, did not support growth unless 50% (by
volume) sterile filtered supernatant of anaerobically grown E.
casseliflavus was added to these cultures. This addition resulted
M boric acid, 20 nM CuCl2䡠2H2O, 0.2 M NiCl2䡠6H2O, 0.3 M
in growth and dechlorination activity, hence allowing isolation
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Na2MoO4䡠2H2O), salts (0.2 mM MnCl2䡠4H2O, 0.5 mM Na2SO4, 1 mM
CaCl2䡠2H2O, 7 mM KCl, 16 mM NaCl, 2 mM MgCl2䡠6H2O, 5 mM NH4Cl, 2 mM and physiological characterization of the curved rod, desig-
KH2PO4), cysteine䡠HCl (1 mM), resazurin (2 mg/liter), and NaHCO3 (50 mM). nated strain DCA1. No contaminating bacteria could be de-
After enrichment and isolation, suitable electron donors (40 mM) for strain tected after cultivation in roll tubes containing 0.1% YE, 40
DCA1 were H2 (40 mM, nominal concentration), formate, and lactate. Media mM pyruvate, or 40 mM glucose or after aerobic cultivation on
with H2 or formate additionally contained 5 mM acetate (carbon source). Re-
placing YE by a defined mixture of amino acids (R7131 RPMI 1640; Sigma-
brain heart infusion agar plates.
Aldrich, Bornem, Belgium) and supplying 1 M vitamin K1 or vitamin K2 (stock Formate could replace H2 as an electron donor in the pres-
solution of 5 mM in pure isopropanol) allowed indefinite subcultivation of strain ence of acetate. However, acetate alone did not support 1,2-
DCA1 under completely defined conditions. DCA dechlorination, indicating that it exclusively served as a
Analysis. Chlorinated substrates and alkenes were identified and quantified by
carbon source. In addition, lactate could be used as electron
headspace analysis or, after extraction in hexane, by gas chromatography with a
flame ionization detector (Chrompack 9002) and a CP-SIL 5CB-MS capillary donor and carbon source, making the addition of acetate un-
column (50 m by 0.32 mm; 1.2-m stationary phase) and with a mass spectrom- necessary. Inorganic electron acceptors that sustained growth
eter (Varian 3400; Magnum Finnigan). Stereochemical conversion products of of strain DCA1 were SO32⫺, S2O32⫺, and NO3⫺. No growth
2,3-dichlorobutane (DCB) stereoisomers were additionally analyzed with a Var- was observed with the electron acceptors SO42⫺, NO2⫺, CO2,
ian 3800 gas chromatograph with Poraplot Q column (25 m by 0.53 mm; 20-m
stationary phase) and a thermal conductivity detector. Inorganic anions were
and fumarate. The supernatant of the E. casseliflavus culture
quantified on a Dionex ion chromatograph with an AS9-HC column (9 mM was strictly required for growth of strain DCA1 with any elec-
Na2CO3 eluent). tron acceptor but could not be replaced by YE, amino acids,
Phylogenetic data. The 16S rRNA genes from a DNA extract (2) of a 50-ml 100 M Fe2⫹, water-soluble vitamins, or volatile fatty acids.
pure culture were amplified, purified, and sequenced by PCR (12) using primers
To identify the unknown excretion product, other Entero-
16F27 (forward) and 16R1522 (reverse) with hybridizing positions 8 to 27 and
1541 to 1522, respectively, referring to Escherichia coli 16S rRNA gene sequence coccus strains such as E. casseliflavus LMG 12901, Enterococ-
numbering (12). Phylogenetic analysis was performed using the Bionumerics cus faecalis LMG 11207, and Enterococcus avium LMG 10774
software package (Applied Maths) after including the consensus sequences in an were additionally tested. Interestingly, the supernatant of the
alignment of small ribosomal subunit sequences collected from GenBank. Com- first two of these strains allowed growth of strain DCA1, while
parison of the last 1,467 nucleotides of the amplified product of the 16S ribo-
somal DNA (rDNA) of strain DCA1 (accession no. AJ565938) with the se-
that of the E. avium strain did not. It has been reported that
quences currently available from the EMBL database (Heidelberg, Germany) both E. casseliflavus and E. faecalis produce menaquinones, in
revealed a 96.6% similarity to the closest relative, Desulfitobacterium hafniense contrast to E. avium (5). In our tests, indeed, 1 M vitamin K1
DCB-2 (DSM 10664; type strain). Multiple alignment was calculated with an or vitamin K2 could replace the Enterococcus supernatant and
open gap penalty of 100% and a unit gap penalty of 0%. A tree was constructed
supported growth and dechlorination. In addition, a defined
by the neighbor-joining method (analyzed and interpreted by the BCCM/LMG
Bacteria Collection, Ghent, Belgium). mixture of amino acids could replace the YE. Pure cultures of
strain DCA1 could be subcultured indefinitely in these com-
pletely defined growth media with S2O32⫺ or 1,2-DCA as the
RESULTS AND DISCUSSION
sole electron acceptor. It would be interesting to investigate if
Initially, we focused on enrichment experiments with 1,2- menaquinones are also essential for growing other dechlorina-
DCA as the sole electron acceptor (besides CO2 present in tors, such as Dehalococcoides and Dehalobacter strains that are
these media). Therefore, a bacterial inoculum was transferred difficult to cultivate (13, 14, 16, 22, 23).
from soil that had been polluted with 1,2-DCA since 1970. When we added 1,2-DCA, acetate, and 0.01% YE to media
After nine transfers with H2 as the electron donor and acetate containing 1 M vitamin K2, growth and dechlorination started
as the carbon source, an unusually high 1,2-DCA dechlorina- but rapidly ceased (Fig. 2). The addition of an electron donor,
tion rate of 1.3 mM/day was achieved in the absence of metha- such as H2, formate, or lactate, to these media sustained
nogenesis and acetogenesis (no CO2 consumption). Stoichio- growth and supported dechlorination of up to 6 mM (cumu-
metric amounts of ethene and HCl were produced. lative) 1,2-DCA. YE was not required for growth and dechlo-
We started isolation on agar (3%, wt/wt) media in roll tubes rination but stimulated both and could be replaced by amino
containing H2 as the electron donor and 1,2-DCA as the elec- acids. Since H2 oxidation and 1,2-DCA reduction cannot be
tron acceptor. About 3% of all transfers of single colonies to coupled to substrate level phosphorylation, the utilization of
liquid media containing H2, 1,2-DCA, and 0.02% (wt/wt) YE these substrates as sole energy sources in defined media indi-
resulted in dechlorination activity. Surprisingly, these dechlo- cates energy conservation via dehalorespiration in strain
rinating cultures contained two different types of bacteria: a DCA1. The 1,2-DCA dechlorination rate exceeded 350 nmolVOL. 69, 2003 DEHALORESPIRATION WITH VICINAL DICHLORINATED ALKANES 5645
dehalorespiring bacteria currently isolated because it neither
dechlorinates any unsaturated chlorohydrocarbons nor carries
out any reductive hydrogenolysis; strain DCA1 exclusively di-
chloroeliminates its saturated chlorosubstrates. This highly se-
lective, fast, and energy-conserving dichloroelimination has
not yet been described for a bacterial isolate in defined media,
which suggests that the dichloroelimination mediated by strain
DCA1 occurs via a novel type of biochemical reaction mech-
anism. The defined growth conditions of strain DCA1 and
further research on its dehalogenase may allow the character-
ization of the currently unexplored respiratory dichloroelimi-
nation biochemistry.
More highly chlorinated alkanes such as hexa-, penta-, and
tetrachloroethanes were not dichloroeliminated. However,
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1,1,2-trichloroethane still supported growth producing VC, il-
lustrating the exclusive dichloroelimination mechanism and
suggesting that the catalytic center of the dehalogenase might
be hindered when more than three chlorine substituents are
present on both vicinal carbon atoms. Consistent with the
exclusivity for dichloroeliminations, no chlorinated methanes,
monochloroalkanes, or nonvicinal dichloroalkanes were de-
chlorinated. This illustrates a high substrate specialization,
which is typical for most dehalorespiring bacteria. All sub-
strates were tested at nontoxic levels. To ensure this lack of
toxicity, cultures that did not start dechlorination of the sub-
FIG. 2. H2 and formate as electron donors for the dechlorination strates after 14 days were additionally supplemented with 400
of 1,2-DCA, as reflected in the corresponding ethene production
(A) and bacterial protein increase (B). Acetate (5 mM) was added in
M 1,2-DCA. In all cases, direct conversion of 1,2-DCA and
all media as a carbon source. The initial concentration of yeast extract growth with this substrate occurred.
was 0.01% (wt/vol). Measurements are average values of triplicate Phase-contrast light microscopy and scanning electron mi-
experiments. In identical media without H2 or formate, growth and croscopy of the motile gram-positive cells of strain DCA1
dechlorination ceased after about 700 M 1,2-DCA conversion. 1,2- revealed curved rods 0.5 to 0.7 m in diameter and 2 to 5 m
DCA was respiked after complete conversion to ethene, leading to a
cumulative nominal concentration of 4.8 mM (all five spikes) in media in length. Occasionally, cell lengths exceeding 10 m were
containing H2 or formate and to 1.2 mM (first two spikes) in media observed. Optimal pH and temperature were 7.2 to 7.8 and 25
without an additional electron donor. to 30°C, respectively. Reductive dechlorination occurred be-
tween 12 and 32°C.
Comparative analysis of the almost complete 16S rDNA
of chloride released per min per mg of total bacterial protein, sequence of strain DCA1 with the sequences currently avail-
while the protein yield was 0.41 ⫾ 0.08 g (mean ⫾ standard able from the EMBL database revealed a 96.6% similarity to
deviation; n ⫽ 12 cultures) of total bacterial protein per mol of the closest relative, Desulfitobacterium hafniense strain DCB-2
chloride released. (DSM 10664; type strain). Physiologically, strain DCA1 is
In addition to 1,2-DCA, a defined range of other chlorinated clearly different from other Desulfitobacterium species, since
alkanes sustained growth of strain DCA1, including 1,2-D, chloroethenes and chlorophenols were not converted, while no
1,2-DCB, D-2,3-DCB, L-2,3-DCB, and meso-2,3-DCB. All other Desulfitobacterium species has been reported to mediate
these substrates (400 M) were completely dechlorinated to dichloroelimination of vicinal dichloroalkanes. One isolate,
the corresponding alkenes with at least 99.9% molar selectiv- Desulfitobacterium sp. strain Y51, is able to partially dechlori-
ity. Final concentrations of the substrates dropped below 2 nate hexa-, penta-, and tetrachloroethanes, in addition to par-
M. The toxicity limit of 1,2-DCA was found to be 1.5 mM tial polychloroethane dechlorination (26). This anaerobe uses
during exponential growth. After six consecutive 5% (by vol- both reductive hydrogenolysis and dichloroelimination reac-
ume) transfers on media containing S2O32⫺ (10 mM) as the tions, which are slower and which produce chlorinated end
sole electron acceptor, strain DCA1 immediately started to products (dichloroethenes). Strain Y51 and strain DCA1 do
dechlorinate vicinal dichloroalkanes when transferred to me- not have any chlorosubstrate in common and show a 16S
dia containing these compounds. Since preculturing strain rDNA similarity of 96.0%. Strain DCA1 has been deposited in
DCA1 with S2O32⫺ or dichloroalkanes as the sole utilizable the BCCM/LMG Bacteria Collection (accession no. P21439)
electron source did not influence its subsequent dechlorination and has been proposed as the type strain of the new species
behavior, these experiments indicate a constitutive dechlorina- Desulfitobacterium dichloroeliminans.
tion activity. Strain DCA1 is not extremely oxygen sensitive, surviving
VC and monochloroethane were not used as electron accep- aerobic conditions for at least 24 h, even though anoxic con-
tors, nor were they formed during 1,2-DCA dechlorination, ditions (lower than ⫺180 mV) were essential for 1,2-DCA
indicating that neither of the compounds is a free intermediate dechlorination. Vitamin B12 (40 nM) was required as a growth
of the process. In that sense, strain DCA1 is different from all supplement for significant dechlorination activity of strain5646 DE WILDEMAN ET AL. APPL. ENVIRON. MICROBIOL.
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FIG. 3. Suggested anti dichloroelimination mechanism catalyzed by strain DCA1 based on the stereoselective production of E- and Z-2-butene
from the meso and DL stereoisomers of 2,3-DCB, respectively. Products formed result from the dichloroelimination of transantiparallel chlorine
substituents on the saturated carbon chain. This position is always present in the most stable conformers. Newman projections are left view for meso
and L isomers and right view for the D isomer.
DCA1. Propyl iodide (50 M) completely inhibited dechlori- to groundwater during the last decades rather suggests a rapid
nation in the dark; the inhibition could be reversed by illumi- dehalogenase development in response to environmental con-
nation. These findings provide strong evidence for the involve- tamination.
ment of a corrinoid in the dichloroelimination reaction (19, The unraveled nutritional requirements of strain DCA1 al-
20). lowed cultivation of the organism at 100-liter scale in pure
In vivo studies with three different 2,3-DCB stereoisomers as culture. In situ pilot tests showed that the addition of 1 g (dry
electron acceptors were performed (Fig. 3). The meso stereo- weight) of cells of strain DCA1 to 1 m3 of groundwater con-
isomer was selectively converted to E-2-butene, while both D taining 1,2-DCA resulted in a complete detoxification within 1
and L enantiomers produced Z-2-butene. Interestingly, in the week. Hence, dichloroelimination of dehalorespiring bacteria
most stable staggered conformational isomers (conformers) of can be considered a highly efficient and unique remediation
the meso compound and both DL compounds, both chlorine tool.
atoms are opposite to each other (transantiparallel position). We presented the isolation, characterization, and growth
Hence, the product pattern indicates a stereoselective anti medium definition of a dehalorespiring bacterium that selec-
dichloroelimination. The less-stable eclipsed meso and DL con- tively and completely dichloroeliminates several vicinal dichlo-
formers, rarely observed below 303 K (30°C), would require a rinated alkanes, including 1,2-DCA and 1,2-D. Besides envi-
syn dichloroelimination on vicinal adjacent chlorine atoms, ronmental and stereoselective fine-chemical applications, this
resulting in the opposite product formation (except for the discovery may contribute to the understanding of respiratory
highest-energy eclipsed DL conformers). Moreover, the high dichloroelimination biochemistry and evolutionary dehalore-
dechlorination rate, comparable with those for dehalogenases spiration. The methods described in this report and the key
catalyzing reductive hydrogenolysis reactions, suggests that the role of menaquinone in the metabolism of strain DCA1 may
most probable conformer is the main substrate of the dehalo- help to isolate other dehalorespirers that perform a complete
genase of strain DCA1. To our knowledge, so far no stereo- dechlorination.
selective anti dichloroelimination of vicinal dichloroalkanes in
mild aqueous conditions at ambient temperatures has been ACKNOWLEDGMENTS
reported (3, 4, 6, 10, 24). We acknowledge fellowships from the European Science Founda-
The exclusive anti dichloroelimination of strain DCA1 tion (Gpoll) and Bijzonder Onderzoeksfonds (S.D.W.); funding by the
makes its dechlorination biochemistry distinct from that of all Deutsche Forschungsgemeinschaft, the EC, and the Fonds der Che-
mischen Industrie (G.D.); and funding by Ghent University (H.V.L.
other known dehalorespiring isolates. The evolution of the and W.V.).
dehalogenase catalyzing this reaction is enigmatic. Natural Christof Holliger and Alexander J. B. Zehnder are acknowledged
production of trace amounts of strain DCA1’s chlorosubstrates for a critical review of the manuscript.
is not likely to provide sufficient selective evolutionary pressure Patents are pending on this technology.
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