Appl Microbiol Biotechnol (2001) 56:491495

DOI 10.1007/s002530100659

O R I G I N A L PA P E R

R. Hauck L. Adrian P. Wendler M. Amidjojo

W. Hegemann H. Grisch

Transformation of 2,2-dichlorodiisopropyl ether in mixed

and pure culture
Received: 2 November 2000 / Received revision: 7 February 2001 / Accepted: 9 February 2001 / Published online: 26 June 2001
Springer-Verlag 2001

Abstract An aerobic enrichment culture derived from a

groundwater contaminated with organic and chloroorganic compounds was adapted to the transformation of
2,2-dichlorodiisopropyl ether (DDE) in a continuous
fixed-bed bioreactor. Continuous DDE removal efficiencies over 90% were achieved with a model water containing 3.3 mM methanol as co-substrate at DDE loading
rates of up to 150 mol l1 day1 with a hydraulic retention time of 24 h. In batch cultures, a stoichiometric release of 2 mol chloride per mol DDE transformed was
observed. From the mixed culture, a strain was isolated
that is able to grow on DDE as the sole energy and carbon source, tolerating DDE concentrations of up to
1 mM. Based on 16S rRNA sequencing, the strain is affiliated with the genus Rhodococcus.

(5.2 M) from the Ohio River (IARC 1986). It was

concluded that DDE is chemically stable in aqueous media and is not biodegraded in river water (NTP 1982).
Kawamoto (1990) found that DDE was also stable for an
experimental period of 40 days in microbial cultures inoculated with aerobic or anaerobic bacteria. Patterson
and Kodukula (1981), however, described a mixed culture aerobically transforming 29 M DDE. No transformation products were detected.
Here we describe an aerobic fixed-bed bioreactor
transforming DDE. From this reactor, a bacterial strain
was isolated that transformed DDE with the concomitant
stoichiometric release of chloride.

Material and methods

Introduction

Chemicals

The toxic and carcinogenic compound 2,2-dichlorodiisopropyl ether (DDE) [bis(2-chloro-1-methylethyl)ether, bis(-chloro-isopropyl)ether] has been widely
used as an extractant and solvent in the chemical industry and as a nematocide (IARC 1986). It is also formed
as a major by-product (35% w/v) of the industrial synthesis of 1,2-propylene oxide and epichlorohydrin (ECH)
(Plinke et al. 1994).
DDE has been detected in effluents from industrial
plants, in several rivers Ohio (3.2532.53 M),
Kanawha and Mississippi Rivers, USA, and the Rhine
and Scheldt Rivers, The Netherlands and in tap water

A crude mixture of DDE was supplied by Dow (Stade, Germany)

as a distillation product from a propylene oxide production process. The crude DDE mixture was treated with sulfuric acid (96%
v/v) and extracted five times with pentane. After neutralization
with 30% (w/v) of NaOH, the ether separated as an oily liquid.
The residual DDE in the aqueous phase was extracted with pentane, combined with the etheral layer, dried with Na2SO4, and filtered. Then, pentane was removed by evaporation (500 mbar,
30 C). The ether was purified by fractional distillation. DDE was
obtained as a colorless liquid (boiling point 7072 C at 12 mbar)
with a purity of 99.5%, determined by gas chromatography.
All other chemicals used were of p.a. grade and purchased
from Merck (Darmstadt, Germany), Sigma (Deisenhofen, Germany) or Aldrich (Deisenhofen, Germany). Silica Bio-Gel P-100 was
obtained from BioRad (Mnchen, Germany).

R. Hauck M. Amidjojo W. Hegemann

Institut fr Technischen Umweltschutz,
Fachgebiet Siedlungswasserwirtschaft, Sekr. KF 7,
Strasse des 17. Juni 135, 10623 Berlin, Germany
L. Adrian () P. Wendler H. Grisch
Institut fr Biotechnologie,
Fachgebiet Technische Biochemie, Sekr. GG1,
Seestrasse 13, 13353 Berlin, Germany
e-mail: lorenz.adrian@tu-berlin.de
Tel.: +49-30-31427509, Fax: +49-30-31427581

Medium preparation
The mineral salt medium used for continuous operation of the
fixed-bed reactor contained (per liter): Na2HPO42 H2O, 2.66 g;
KH2PO4, 1.36 g; (NH4)2HPO4, 0.45 g; Mg(HCO3)2, 0.12 g. Trace
metals were added resulting in final concentrations of (per liter):
MnSO4H2O, 1.5 g; H3BO3, 50 g; ZnSO4, 50 g; CaCO33 H2O,
1750 g, Na2MoO42 H2O, 1.5 g; CoCl26 H2O, 50 g; NiCl2
6 H2O, 5 g; FeSO4, 500 g. The pH was adjusted to 7.0 to 7.1.
The medium was autoclaved before organic substrates were added.

492
(Ox 1100, Schott) with electrodes integrated in the reactor headspace. Data collection and regulation of the oxygen dosage were
carried out using Dasylab software (version 3.00.10, Datalog,
Mnchengladbach, Germany). Dissolved oxygen concentration
was held constant at 5 mg l1. The pH was held between 7.0 and
7.1.
Batch cultivation

Fig. 1 Configuration of the aerobic fixed-bed bioreactor

For batch cultivation a mineral medium described by Janssen

et al. (1988), was used and DDE was added to final concentrations
between 0.25 to 1 mM. The medium was stirred for at least 24 h
prior to inoculation.

Batch cultivation was done in injection flasks with a total volume

of 120 ml. Twenty ml of sterile medium containing different carbon sources were added to each culture flask. The flasks were
closed with Teflon-lined rubber septa and aluminum crimp caps.
Syringes were used for the transfer of 2% (v/v) inocula. All flasks
were incubated at room temperature in the dark on a rotary shaker
at 120 rpm.
For isolation of DDE-transforming bacteria, single colonies
were repeatedly subcultured on Luria-Bertani (LB) agar plates
containing 10 g l1 trypone, 5 g l1 yeast extract, 10 g l1 NaCl and
1.5% agar (Roth, Karlsruhe, Germany) at pH 7.0. Strain DTB was
deposited at the German Collection of Microorganisms and Cell
Cultures (DSMZ, Braunschweig, Germany) under the number
DSM 44534.
Different carbon sources were tested with a GN-Microplate assay (Biolog, Hayward, USA) according to the manufacturers instructions, using cell suspensions of 3108 cells ml1 grown on
liquid LB medium without DDE. To test for growth on DDE as
carbon source, silica Bio-Gel P100 was used for gel formation
(Funk and Krulwich 1964). A 2-l drop of DDE was added onto a
sterile filter disk that was placed in the middle of a plate homogeneously inoculated by a bacterial suspension. The gel plates were
incubated with DDE in a closed vessel to avoid DDE evaporation.

Inoculum
Analytical methods
A sample of 25 ml of groundwater from the vicinity of a chemical
production site in Berlin, Germany, contaminated with different
chloroorganic and organic compounds, including benzene, 1,2dichloropropane (DCP), diethyl ether, mono- and dichlorobenzene, was used as inoculum. The sample was mixed with 125 ml
of mineral medium without additional substrates. The sample was
transferred to a 500-ml flask and closed with a Teflon-coated rubber septum. After incubation for 5 days at room temperature on a
rotary shaker at 126 rpm, turbidity indicated bacterial growth,
and 80 ml of porous Siran glass (type 012, Schott, Hofheim am
Taunus, Germany) were added. Every 5 days, the carrier material
was transferred to a fresh mixture of 25 ml groundwater and
125 ml mineral medium. After six transfers, the porous Siran glass
was overgrown with a biofilm, which was mixed with 220 ml of
fresh Siran glass and transferred to a 5-l reactor. The reactor
(Fig. 1) was continuously operated with mineral medium initially
containing DCP (488 M), methanol (0.62 mM), benzene
(0.41 mM), monochlorobenzene (14 M), propanol (33 M), and
diethyl ether (13.5 M) as carbon source but not DDE. Within a
period of 13 months, the organic substrates were changed to a
composition of 26.5 M DCP, 1.25 mM methanol, 0.69 mM benzene, 83 M propanol, and 13.5 M diethyl ether. To this medium,
15 M DDE was finally added to investigate DDE transformation.
Fixed-bed reactor cultivation
Complete mixing was realized by pumping the liquid upward
through the fixed-bed reactor, establishing a well-mixed liquid
phase (Fig. 1). Fresh medium was supplied continuously using a
Teflon-coated membrane pump (gamma 4/W, Prominent, Heidelberg, Germany) from a 20-l feeding tank. All connections and fittings were made either from glass, Teflon or Viton. The temperature was maintained at 2527 C. The reactor performance was
monitored by measuring pH (Inlab 417, Mettler-Toledo, Urdorf,
Swiss), temperature (W 2130, Schott) and oxygen concentration

DDE was quantified by gas chromatography using flame ionization detection. Probes were injected either directly from the gas
phase using a headspace autosampler (HS 40, Perkin Elmer, Norwalk, USA) combined with a gas chromatograph (GC) HP 5890
series II (Hewlett Packard, Palo Alto, USA) or after extraction
with hexane. For headspace analysis, 5-ml samples were transferred to headspace vials, diluted with 15 ml deionized water,
sealed, and treated with ultrasound for 20 min. Sample heating
time was 40 min; the temperatures of the sample, injection needle
and transfer capillary were 80, 100 and 125 C, respectively. After
pressurization (3 min) and injection (0.1 min), the withdrawal time
amounted to 0.2 min. The GC was equipped with a 25 m
0.32 mm capillary column CP-Sil 5 CB with a 1.2-m stationary
phase (Chrompack, Middelburg, The Netherlands). Helium
99.999% (v/v) was used as carrier gas. Inlet and detector temperatures were 200 and 300 C, respectively. The initial oven temperature was held at 35 C for 3.5 min and then increased to 220 C
with a rate of 10 C min1. Hexane extraction of DDE and ECH
from aqueous solutions and analysis by gas chromatography of the
extracts was done as described for chlorobenzenes (Adrian et al.
1998).
Chloride concentrations were determined by ion chromatography (DX-120, Dionex, Idstein, Germany) of the culture supernatants after centrifugation (10,000 g, 20 min). Growth of organisms
was determined photometrically at 578 nm.
The 16S rRNA gene sequence was determined from a colony
that was suspended in water and heated to 98 C for 10 min. For
PCR, the primers 616V (5-AGA GTT TGA TC/TA/C TGG CTC
AG-3) and 1492R (5-CGG C/TTA CCT TGT TAC GAC-3)
were used at a hybridization temperature of 55 C (Adrian et al.
2000). The amplicon was cleaned (QIAquick PCR purification kit,
Qiagen, Hilden, Germany) and directly sequenced using 616V,
1492R and four internal primers (Adrian et al. 2000). The sequence of strain DTB was deposited at the NCBI database under
the accession number AY024343.

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Results
Transformation of DDE in a fixed-bed reactor
To establish an aerobic DDE-transforming mixed culture,
a continuous fixed-bed reactor was used that previously
showed high transformation potential towards a number
of different chloroorganic compounds (Meierling 1998).
A mineral medium already containing DCP 26.5 M,
benzene 0.69 mM, methanol 1.25 mM, 2-propanol
83 M, and diethyl ether 13.5 M was amended with
15 M DDE and fed into the reactor containing Siran
glass covered with microorganisms (Fig. 1).
During the first 47 days of reactor operation, the DDE
concentration in the effluent was as high as the concentration in the feed solution, demonstrating that stripping
effects could be neglected (Fig. 2). At a stable DDE
influent concentration of 15 M (space loading=
4.8 M days1), the effluent concentration decreased beginning at day 47. Within the next 30 days, the transformation of DDE rose steadily to over 90%. During the
successive increase of the DDE concentration in the influent to 47 M and the stepwise reduction of the hydraulic retention time (HRT) to 24 h, DDE transformation remained stable between 97 and 100%.
During reactor operation, the number of substrates
was reduced by stopping the addition of DCP, propanol
and diethyl ether. From day 96 on, only methanol
(0.41 mM) and benzene (0.93 mM) were supplied.
In additional fixed-bed reactor experiments, the influent concentration of DDE was further increased. With
2.34 mM methanol as the only cosubstrate, a DDEspace loading of up to 147.8 M day1 at an HRT of 24 h
still resulted in a DDE transformation of 98% (data not
shown).

Fig. 2 Aerobic transformation of 2,2-dichlorodiisopropyl ether

(DDE) in the continuous fixed bed reactor. HRT Hydraulic retention time, DDE influent concentration, DDE effluent concentration, DDE transformation (%)

Fig. 3 Remaining DDE concentrations after 7 days of incubation

in batch cultures of the enriched consortium containing different
initial concentrations of methanol. Shown is the mean of triplicate
culturesSD

Batch cultivation experiments

Inoculum from the reactor was used for enrichment and
isolation of the DDE-transforming bacteria in batch culture. In mineral medium containing 250 M DDE and
5 mM methanol as co-substrate, the enriched culture
transformed DDE completely within 7 days after inoculation (Fig. 3). DDE transformation was also observed at
lower concentrations of methanol; however, in the absence of methanol the transformation was significantly
slower. Control bottles without inoculum did not transform DDE (Fig. 3). DDE concentrations also did not decrease when autoclaved inocula were used (data not
shown).
Other compounds present in real waste water from the
industrial production of propylene oxide were tested for
their effect onto DDE transformation by the enriched
culture. DDE transformation was increased significantly
by 5 mM 1,2-propanediol (Fig. 4). However, the addition
of 5 mM ECH, 1,2-dichloropropane or diisopropyl ether
inhibited DDE transformation. Incubation of the mixed
culture with DDE together with ECH led to a stoichio-

Fig. 4 Remaining DDE concentrations after 7 and 14 days in

batch cultures of the enriched consortium amended with different
co-substrates (5 mM) potentially present in real waste waters. Results are given as the mean of triplicate culturesSD

metric release of chloride from ECH, while DDE was

not transformed. In contrast, no chloride was released
from 1,2-dichloropropane. Simultaneous addition of ethanol, 1-propanol, and 2-propanol (5 mM each) did not
significantly alter the rate of DDE transformation
(Fig. 4).
DDE transformation was observed in batch cultures
of the enriched consortium with up to 1 mM initial DDE
concentration (data not shown). The maximum concen-

494

Fig. 6 DDE transformation by strain DTB, starting with different

initial DDE concentrations from 30 to 1000 M but without methanol as a co-substrate. 2% (v/v) inocula were used. Residual DDE
concentrations are given as means of triplicate culturesSD

Fig. 5ac Change of DDE concentration, chloride concentration,

and pH in batch cultures of the enriched consortium initially containing 250 M DDE and fed with a further 500 M DDE at day 7
and 250 M DDE at day 14. The 500 M added at day 7 only
slowly dissolved between day 7 and day 14. Media were inoculated with 2% (v/v) inocula containing about 6105 DDE-transforming cells. Negative control: 250 M DDE, no methanol, no inoculum; 250 M DDE, no methanol; 250 M DDE, 5 mM
methanol; no DDE, no methanol; no DDE, 5 mM methanol

tration that was transformed within 7 days in cultures

containing 5 mM methanol as a co-substrate was about
450 M. Fed-batch experiments demonstrated a stoichiometric release of 2 mol chloride per mol DDE transformed (Fig. 5A, B). Concurrent with the release of chloride, a decrease of the pH from 7.0 to 5.76.1 was detected (Fig. 5C). A decrease in pH was only observed in
inoculated cultures containing DDE.
Strong inhibition of DDE transformation was observed in the presence of CuSO4. While without CuSO4
250 M DDE was completely transformed after 14 days
of incubation, cultures amended with 4.8 M CuSO4 still
contained DDE concentrations of 200250 M after
14 days. Under these conditions, DDE concentrations
decreased only slowly after prolonged incubation for
several weeks.
Isolation of a DDE-transforming strain
By most probable number determination, the number of
DDE-transforming bacteria in the enriched culture was
estimated to be 1.5106 cells ml1, representing more
than 10% of the total population. Subsequently, isolation
was achieved by repeated transfers of single colonies to
fresh agar plates. Several isolated strains were tested for
DDE transformation in liquid culture. One isolate, designated strain DTB, transformed DDE at a rate comparable

to the mixed culture and was selected for further characterization. All tested DDE concentrations up to 1000 M
were transformed by the isolate within 3 weeks of incubation (Fig. 6). No toxic effects of high DDE concentrations were found. Strain DTB also transformed DDE in
the presence of 1% sodium chloride (data not shown).
Colony formation of strain DTB after 3 weeks of incubation was detected on mineral medium solidified with silica only when DDE was present. Other carbon sources
used included -D-glucose, D-fructose, D-galactose, glycogen, citrate, D,L-lactats, succinate, L-asparagine, L-phenylalanine, L-proline, and L-serine. The cells of strain
DTB were short rods, 31 m in size, and nonmotile.
The 16S rRNA gene, comprising 1421 bp, showed a sequence identity of 100% to the 16S rRNA gene sequences of Rodococcus erythropolis (formerly Nocardioides
simplex; GenBank accession number U81990; Yoon
et al. 1997), Rhodococcus sp. strain SRB1948-A07
(GenBank accession number AB010911, Colquhoun et
al. 1998), and Rhodococcus erythropolis isolate EK5
(GenBank accession number AJ237967, Katsivela et al.
1999).

Discussion
In the present study, a continuous culture was established
in a fixed-bed bioreactor almost completely transforming
space loads of up to 147 M day1 of DDE. The culture
tolerated varying DDE concentrations, co-substrate concentrations, and changing reactor-operating conditions.
This stability could be important if the bacterial culture
is used for biological treatment of real waste waters, e.g.,
from the production of propylene oxide or ECH. The
high stability of the culture in the fixed-bed reactor
might partly be due to the immobilization, since immobilized microorganisms are often more stable against
changes in substrate concentrations, high salt concentrations or other suboptimal growth and degradation conditions (Rehm 1990). Siran glass as a carrier material was
chosen because it provided a high surface area for bio-

495

film formation but did not show accumulation of DDE

on the surface, which could lead to toxic concentrations
(Kuhlmann 1999).
Batch cultivation of the DDE-transforming mixed
culture and isolation of a DDE-transforming strain allowed a more detailed physiological characterization of
the organism. No transformation of DDE occurred at
NaCl concentrations higher than 1%, which might be a
drawback in using the strain for remediation, because
waste water from the synthesis of propylene oxide and
ECH usually contains high salt concentrations. However,
the significant increase of DDE transformation in the
presence of methanol or 1,2-propanediol might be advantageous in waste water treatment.
The mixed culture and the pure strain DTB transformed DDE also at substantial rates in a defined medium without any co-substrates. A number of different
mechanisms of microbial ether transformation have been
described, including oxygenative cleavage via monooxygenases, oxidative and reductive mechanisms, direct hydrolysis of the C-O bond, carbon-oxygen lyase-mediated
cleavage, and anaerobic cleavage of methyl-aryl ethers
(White et al. 1996). Rhodococcus spp. have previously
been shown to degrade cyclic ethers (Bernhardt and
Diekmann 1991). Wilkes et al. (1996) demonstrated that
Sphingomonas sp. strain SS33 was able to grow with
4,4-dichlorodiphenyl ether as carbon source. The ether
was mineralized under release of 93% of the chlorine
substituents as chloride ions with the intermediary production of 4-chlorophenol and 4-chlorocatechol.
A stoichiometric release of chloride from DDE and
the change in the pH in batch experiments demonstrated
that both chlorine substituents were removed from DDE
by strain DTB. Complete mineralization of DDE by
strain DTB has not been shown so far; however, growth
of the isolate in a purely synthetic medium with DDE as
the only carbon and energy source, as well as growth to
colony size on silica gel plates only if DDE was added,
demonstrated that DDE can be used as a growth substrate.
Acknowledgements This study was carried out within the framework of the co-operative research program Sonderforschungsbereich 193 Biological treatment of industrial wastewater, supported by the Deutsche Forschungsgemeinschaft. We thank W.
Rotard for his advice on DDE purification.

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