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Bioutilization of thiodiglycol, the product of mustard detoxification:isolation of degrading strains, study of biodegradation process and
metabolic pathways
I.T. Ermakova, I.I. Starovoitov *, E.B. Tikhonova, A.V. Slepen’kin, K.I. Kashparov,A.M. Boronin
G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
Received 8 January 2002; accepted 12 February 2002
Abstract
Alcaligenes xylosoxydans subsp. denitrificans TD1, possessing degrading activity against thiodiglycol (TDG), was isolated from
soil samples contaminated by the products of mustard detoxification. Using long-term selection, the most active strain A.
xylosoxydans TD2 was obtained. The effect of cultivation conditions*/pH, specific substrate loading (SSL) and substrate
concentration on the efficiency of TDG destruction process were determined. The initial microbial attack on the TDG molecule
involved oxidation of both sulphur atom and primary alcohol groups with the formation of diglycolsulphoxide (DGSO) and
thiodiglycolic acid (TDGA), respectively. The transformation to DGSO is a catabolic deadlock since this compound is not oxidized
by bacterial cells or used by them as a sole carbon source for growth. The key metabolic reaction of TDG degradation is the
uncoupling of the C�/S bond of intermediates*/TDGA and thioglycolic acid (TGA). This reaction leads to the formation of SO42�
ions and acetate, which is involved in the reactions of central metabolic pathways. A scheme for TDG metabolism by A.
xylosoxydans TD2 was suggested. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Mustard; Thiodiglycol; Biodegradation; Metabolism; Transformation; Alcaligenes xylosoxydans
1. Introduction
In accordance with the Chemical Weapons Conven-
tion a concept of two-steps destruction of chemical
warfare (CW) agents has been developed in Russia.
Chemical detoxification is provided at the first step and
elimination of detoxification products at the second
step. The destruction of these products by incineration is
highly expensive since in addition to direct energy
consumption the process requires retaining aerosols
harmful to the environment. Biotechnological methods
are environmentally safe and are an attractive alter-
native to incineration.
Biocatalytic methods for detoxification of organo-
phosphorous CW by uncoupling of P�/O and P�/F
bonds using organophosphate hydrolase have been
developed [1,2]. The search for microorganisms posses-
sing high activity of C�/P lyase which splits C�/P bond of
methylphosphonic acid and its analogues (the products
of enzyme-based hydrolysis of neurotoxic CW agents*/
sarin and soman) was performed [3,4].
Furthermore, an environmentally safe method for the
destruction of mustard�/lewisite mixture has been devel-
oped. This method includes several consequent steps:
chemical detoxification, electrochemical processing of
detoxification products and complete biodegradation of
electrolysis organic products by a selected microbial
association in a fluidized bed reactor [5].
Mustard (bis(2-chloroethylsulphide) is a vesicant,
slightly soluble in water, hydrolyzable in alkaline
medium. A non-chlorinated product of mustard hydro-
lysis is thiodiglycol (TDG) (bis(2-hydroxyethyl)sulphide
[6]. TDG is a stable product, well soluble in water and
less toxic than mustard, however it was included in the
list of CW precursors that should be destroyed in
accordance with the Chemical Weapons Convention.
* Corresponding author. Tel.: �/7-967-733-671; fax: �/7-95-956-
3370.
E-mail address: [email protected] (I.I. Starovoitov).
Process Biochemistry 38 (2002) 31�/39
www.elsevier.com/locate/procbio
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 4 5 - 6
TDG can be a product of hydrolysis of mustard in soil
and will accumulate and remain in nature for long
periods. The development of methods for soil bioreme-
diation by introduction of microorganism-destructors ofTDG in combination with agrotechnical approaches is
an optimal solution for the problem of cleaning-up the
soil contaminated by mustard or its detoxification
products.
Previously, the ability for TDG oxidation has been
studied in more than 150 strains of bacteria, yeasts,
micromyces and actinomyces belonging to 35 different
genera. However, among the investigated organismssuch activity was not found. On the other hand, even
bacteria Pseudomonas , Corynebacterium, Rhodococcus
genera which degrade the chemical analogue of TDG,
diethyleneglycol, did not oxidize TDG itself [7]. Bacter-
ial strains utilizing TDG as sole carbon source were
isolated from mustard contaminated soils by the tech-
nique of culture enrichment after a 9-month incubation
with TDG [8]. One of them identified as Alcaligenes
xylosoxydans subsp. xylosoxydans (SH91) was used to
develop the environmentally safe method of mustard
degradation involving its alkaline hydrolysis followed by
the bioutilization of TDG formed [9,10]. The TDG
degrading strain Pseudomonas sp . 8-2 was also isolated
after long-term incubation of soil samples with TDG
and mineral nutrient components [7]. The bacteria
Rhodococcus rhodochrous IGTS8 utilized TDG as thesulphur source for growth [11].
The identification S-(2-hydroxyethylthio)acetic acid,
TDGA and DGSO among oxidation products of TDG
by A. xylosoxydans (SH91) as well as the above organic
acids in Pseudomonas sp. 8-2 indicates that the initial
steps in the microbial degradation of this compound are
the oxidation of the primary alcohol groups and a
sulphur atom. The pathways of subsequent catabolismof the formed metabolites remain unknown.
The aim of this work was to isolate and select the
microorganisms able to utilize TDG as the sole carbon
source and to study the conditions optimal for the
bioutilization process as well as the mechanism of
oxidation of this compound.
2. Materials and methods
2.1. Microorganisms
Two strains of Gram-negative obligate aerobic bac-
teria A. xylosoxydans subsp. denitrificans that consume
TDG as the sole carbon source were used. Strain TD1was isolated from soil samples contaminated by mustard
detoxification products. Strain TD2 was obtained as a
result of long-term selection of strain TD1.
2.2. Medium and cultivation
The mineral composition of the MS medium for
cultivation was (g/l): NH4Cl�/2.0, MgSO4�/7H2O�/0.2,CaCl2�/6H2O�/0.01, K2HPO4�/10.0, KH2PO4�/1.0 and
microelements (mg/l): FeSO4�/7H2O�/2.5, CuSO4�/
5H2O�/2.0, H3BO3�/0.06, ZnSO4�/7H2O�/20.0,
MnSO4�/1H2O�/1.0, Na2MoO4�/2H2O�/0.3, NiCl2�/
6H20�/0.05.
The carbon sources were TDG (Merck), TGA and
acetate (Reachim), TDGA, DGSO and sulphoacetic
acid (synthesized as described in [12]).Cultivation was achieved in 750 ml flasks, each
containing 100 ml medium on the shaker (220 rpm) or
in the fermenter with automatic supporting of pH and
pO2. The concentration of dissolved oxygen was 0.5 g/l
per h and 30% of air saturation, respectively. pH was
maintained at 7.0�/7.5 by addition of 20% NaOH. The
temperature of incubation was 30 8C.
The inoculum culture was grown on the agar MSmedium with 2 g/l of TDG.
2.3. Growth control
Growth was controlled by the change in optical
density (Specol spectrophotometer) at 560 nm (OD560)
and the number of colony-forming units (CFU). OD560
units was converted to dry biomass weight with a
conversion factor of 0.5 obtained experimentally for A.
xylosoxydans TD2.
2.4. Oxidative activity
To study oxidative activity, the bacterial culture was
grown in the fermenter. Exponentially growing cells
were harvested by centrifugation, twice washed and
resuspended in 50 mM phosphate buffer (pH 7.3).
2.5. Analytical methods
TDG, TDGA, DGSO and TGA concentrations were
determined by HPLC (Model LKB-2150) at 214 nm
(LKB-2151, UV-detector) with a SEPARON SGX C18
column, (3.3 mm�/150 mm, 5 mm). The HPLC running
conditions were as follows: column temperature, 65 8C;mobile phase, 3 mM phosphoric acid in deionized water;
flow rate, 1.0 ml/min. The consumption peaks were
registered by the interfaces of NELSON ANALYTI-
CAL 900 Series and PC Olivetty M-24. The associative
peaks were identified by their comparison with the peaks
of standard compounds. The data were processed by
‘Nelson Analytical’ application. Residence times of the
peaks for TDG, TDGA, DGSO and TGA correspondedto 2.24, 1.54, 1.26, 1.59 min, respectively.
Mass-spectrometric analysis was performed on chro-
mato-mass spectrometer HP-5793 with chromatograph
I.T. Ermakova et al. / Process Biochemistry 38 (2002) 31�/3932
HP 6890, the capillary column length, 50 m; phase,
ULTRA-2. Chromatographic conditions: programmed
heating from 40 up to 250 8C with the rate of 108/min.
The cell respiration was measured in a polarographiccell with platinum Clark-type electrode on polarograph
LP-7 (Chezh Republic).
The concentration of SO42� ions was determined by a
gravimetric method [13].
3. Results
3.1. Isolation and selection of TDG-degrading
microorganisms
TDG-degrading microorganisms were isolated from
soil samples contaminated by mustard detoxification
products. The soil samples were provided by the State
Institute of Technology of Organic Synthesis (Shikhany,
Saratov region, Russia). The moistened soil (up to 40%
of moisture) were first incubated with TDG and mineral
components of MS medium for 3 months at 30 8C and
then for 7 days in liquid MS medium with TDG in flaskson the shaker. Four types of bacteria differing by their
morphological characteristics (type of colony, size and
shape of cells, motility) were isolated on nutrient agar.
However, only one type of isolate grew on MS agar
medium with TDG.
The strains of this type had a long lag-phase and a low
specific growth rate on TDG. One isolate was identified
according to Bergey’s Manual [14] as A. xylosoxydans
subsp. denitrificans TD1. Its lag-phase increased from 48
to 120 h with increasing of TDG concentrations from
0.6 to 3.2 g/l (Fig. 1). At the same time, the specific
growth rate increased from 0.003�/0.005 to 0.01 h�1.
The representatives of other bacterial types isolated
from the mixed culture grew neither on TDG nor on
possible intermediates of its oxidation: TDGA, DGSO,
TGA. Their growth in the MS medium with TDG wasprobably supported by the lysis products of A. xylosox-
ydans strains.
For selection of A. xylosoxydans TD1 the exponential
culture was re-inoculated several times into the medium
containing increasing TDG concentrations from 1 to 3
g/l. Within a 6-month period this method allowed
selection of some variants of culture with a lag-phase
of 4�/8 hours and a specific growth rate of 0.04�/0.045h�1 (Fig. 1).
One of these A. xylosoxydans TD2 was used to study
the relationship between cultivation conditions and
growth characteristics as well as the mechanism of
TDG oxidation.
3.2. Effect of cultivation conditions
3.2.1. pH of the medium
During cultivation of A. xylosoxydans TD2 in the
medium with TDG the pH values decreased from 7.5 to
5.0�/5.5 in 48 h in spite of high phosphate concentrations
(55 mM). Then the growth and substrate consumption
stopped completely (Fig. 2). This culture grew in themedium with glutamate as the sole carbon source at pH
5.2 though with lower specific growth rates than at pH
7.5 (data not shown).
Sterile 20% NaOH solution or CaCO3 were used to
maintain pH in the range of 7.0�/7.5. Under these
conditions the biomass continued to increase till com-
plete consumption of TDG was achieved. Growth
curves plotted in accordance with the results on
Fig. 1. The growth dynamics of A. xylosoxydans TD1 (j, OD560; I,
TDG) and A. xylosoxydans TD2 (m, OD560; k, TDG) in the medium
with TDG.
Fig. 2. The effect of pH on the growth dynamics of A. xylosoxydans
TD2 (j, OD560; I, TDG at pH 7.5�/5.0; m, OD560; m, CFU; k,
TDG at pH 7.5�/7.0).
I.T. Ermakova et al. / Process Biochemistry 38 (2002) 31�/39 33
measurements of OD560 and the number of CFU were
identical (Fig. 2).
The growth of A. xylosoxydans TD2 was accompa-
nied by SO4�2 ions accumulation in the medium. Their
content increased from 0.08 g/l (initial MS medium) to
1.0 g/l at pH 7.5�/5.0 and 2.6 g/l at pH 7.5�/7.0 in the
stationary phase.
3.2.2. Specific substrate loading
The interrelation between SSL at the beginning ofcultivation (g TDG/g biomass) and the duration of the
lag-phase and growth phase, specific growth rate,
biomass yield were studied. The initial cell concentra-
tions were changed between 0.02 and 0.5 g/l at TDG
concentration of 2.5 g/l. The lag-phase with SSL of 125
g/g was 20 h, growth stopped as a result of complete
substrate consumption in 160 h. When SSL decreased to
18 g/g the lag-period decreased to 7 h, the culturepassing to stationary phase in 75 h. These parameters
were equal to 3 and 42 h, respectively, with SSL 5 g/g
(Fig. 3). The maximal specific growth rate and the
biomass yield were independent of SSL. Their values
were 0.037�/0.04 h�1 and 0.28�/0.3 g/g TDG, respec-
tively, for these experiments. The same regularity was
observed when A. xylosoxydans TD2 was cultivated in
the medium with initial TDG concentration 20 g/l (datanot shown).
3.2.3. Initial substrate concentration
Experiments were carried out with initial TDG
concentrations ranging from 1.15 to 39.3 g/l and SSLof lower than 20. Under these conditions the specific
growth rate in the exponential phase had maximal
values of 0.042�/0.045 h�1 in the range of substrate
concentrations of 3.5�/10.0 g/l. The same pattern was
also observed with the biomass yield. The values of these
parameters gradually decreased with further increase in
initial TDG concentration to 20�/25 g/l, but the lag-
phase did not exceed 4 hours. In the medium with 39.3 g/
l of TDG the culture started to grow only in 144 h after
TDG concentration decreased to 25 g/l due to itsoxidation by the cells introduced as the inoculum
culture. The specific growth rate under these conditions
was 0.004 h�1, whereas in the medium with initial TDG
concentration of 26.4 g/l it was equal to 0.015 h�1
(Table 1).
3.3. Metabolism of TDG
The TDG oxidation pathways by A. xylosoxydans
TD2 were studied by means of: (a) analysis of inter-
mediates which accumulate in the growing culture under
different cultivation conditions (pH, SSL, pO2, substrateconcentration) and during TDG oxidation by resting
cells; (b) cultivation in the media with putative inter-
mediates of TDG oxidation*/TDGA, TGA, DGSO,
acetate and sulphoacetic acid as the sole carbon source;
(c) measurement of the cell oxidative activity with the
above compounds.
3.3.1. Accumulation of intermediates
The results of HPLC analysis showed the presence of
DGSO and TDGA in the culture liquid of A. xylosox-
ydans TD2 growing on TDG as the sole carbon source.
These compounds were presumably the products ofbiological TDG oxidation, since they were absent from
the mineral medium with TDG incubated at the same
conditions without bacterial cells or with inactivated
cells. Their maximal amount increased with increase in
initial TDG concentration.
Along with DGSO and TDGA, the concentration of
SO42� ions (containing 80�/90% of sulphur being a part
of the consumed TDG) also increased (Table 1).Chromato-mass-spectrometric analysis of intermedi-
ates of TDG oxidation by intact cells (native and
diasomethane-methylated samples) showed the presence
of thioglycolic acid (1), thiodiglycolic aldehyde (2),
diglycolsulphoxide (3), dimethyl ether of thiodiglycolic
acid (4) and the methyl ether of acetic acid (5) (Table 2).
The accumulation of DGSO could be detected at the
beginning of the exponential growth phase. DGSO thencontinuously increased concomitant with the decrease in
TDG concentration and remained at the same level in
the stationary phase after complete consumption of the
growth substrate (Fig. 4a,b).
The accumulation of TDGA in the medium was
largely dependent on the cultivation conditions. This
acid was released into the external medium during
exponential growth in flasks at pH 7.0�/7.5 reachingthe maximal concentration in the retardation phase and
completely disappeared at the beginning of the station-
ary phase (Fig. 4a). However, only trace amounts of
Fig. 3. The effect of specific substrate loading on the growth dynamics
of A. xylosoxydans TD2 (SSL 125: j, OD560; I, TDG; SSL 18: m,
OD560; ^, TDG; SSL 5, m, OD560; k, TDG).
I.T. Ermakova et al. / Process Biochemistry 38 (2002) 31�/3934
TDGA were detected in the medium in the fermenter at
optimal values of pH and pO2 (Fig. 4b).
High initial TDG concentration (39.3 g/l) inhibited
growth while substrate was still actively oxidized to
TDGA by inoculum cells. The increase of biomass
occurred only when the TDG concentration dropped
to 25 g/l, then the acid concentration reached 6.6 g/l. At
the same time, the rate of acid accumulation decreased
from 0.045 g/l per h in the lag-phase to 0.01 g/l per h in
the growth period. No accumulation of DGSO was
observed in the lag-phase and its concentration in-
creased insignificantly in the growth phase (Fig. 5).
3.3.2. Utilization of intermediates as substrates for
growth and oxidation
The ability of A. xylosoxydans TD2 to assimilate
intermediates of TDG oxidation was investigated.
DGSO is neither utilized as the sole carbon source for
growth, nor inhibits bacterial growth on TDG when
both substrates simultaneously present in the medium.
DGSO accumulated in the medium with TDG only in
growing culture since resting cells grown on TDG did
not oxidize it. Sulphoacetic acid containing as DGSO an
oxidized sulphur atom was neither utilized as carbon
source for growth nor oxidized by cells (Table 3).
Table 1
The effect of TDG concentration on A. xylosoxydans TD2 growth, substrate consumption and intermediate accumulation
TDG concentration (g/l) Biomass (g/l) mmax (h�1) Biomass yield (g biomass/g TDG) Accumulation in medium (g/l)
SO42� DGSO TDGA
1.15 0.27 0.022 0.23 n.d. 0.1 0.005
1.74 0.47 0.03 0.27 n.d. 0.21 0.006
2.6 0.72 0.036 0.28 1.6 0.3 0.014
3.24 0.9 0.042 0.27 n.d. n.d. n.d.
7.46 1.95 0.042 0.26 5.1 0.32 0.21
9.3 2.2 0.045 0.24 n.d. n.d. n.d.
13.9 2.7 0.029 0.19 n.d. n.d. n.d.
15.2 3.4 0.024 0.22 n.d. 0.53 0.52
22.7 3.2 0.018 0.14 n.d. n.d. n.d.
26.4 3.2 0.015 0.12 n.d. n.d. n.d.
39.3 2.4 0.004 0.06 n.d. 0.69 8.8
n.d., no determination was performed.
Table 2
Chromato-mass-spectrometric analysis of intermediates of TDG oxidation by A. xylosoxydans TD2
I.T. Ermakova et al. / Process Biochemistry 38 (2002) 31�/39 35
In contrast to DGSO, TDGA was consumed as a sole
carbon source. The growth characteristics were largely
affected by the concentration of this acid in the medium.
Under optimal cultivation conditions (pH, aeration) the
growth with 0.8 g/l of acid started after a long lag-phase
(72 h) with the specific growth rate of 0.008 h�1.
However it was completely inhibited by 2 g/l of
TDGA. The growth characteristics in the medium with
3 g/l of TDG (lag-phase 4 h, mmax 0.04 h�1) did not
change in the presence of 0.2 g/l of TDGA while the lag-
phase increased to 48 hours and the specific growth rate
decreased to 0.018 h�1 with an acid concentration of 2
g/l (Table 3).TDGA was actively oxidized by resting cells. The
respiration rate reached a maximal value of 172 natom
O2/mg per min at an acid concentration of 10 mM
(Table 3). TGA, acetate and SO42� were identified as the
intermediates of TDGA oxidation under these condi-
tions.
TGA was oxidized by cells with a rate comparable to
that of TDGA oxidation, but it was not utilized as acarbon source for growth. Acetate was actively meta-
bolized under growth conditions and also oxidized in the
absence of growth (Table 3).
4. Discussion
The results of the present work along with available
literature data have shown that representatives of A.
xylosoxydans are able to utilize TDG as the sole carbon
source. Evidently, the population of these bacteria in
soil is heterogeneous and includes strains possessing
different activity against TDG. Long-term selection
allows the isolation of strain A. xylosoxydans ssp.
denitrificans TD2 growing in the media with high
TDG concentrations after short lag-phase with mmax�/
0.04�/0.045 h�1.The efficiency of TDG utilization is affected by pH
value in the medium. The pH drops precipitously due to
of SO42� ions accumulation. The strain A. xylosoxydans
TD2 grows in the medium with glutamate at pH 5.2,
whereas in the presence of TDG growth stops when pH
falls only to 5.5. Similarly TDG oxidation by immobi-
lized cells of A. xylosoxydans ssp. xylosoxydans (SH91)
is also blocked at pH lower than 6.0. [15]. These resultspoint to the significance of pH in TDG metabolism.
There are literature data about the importance of SSL
in the initial period of cultivation for growth dynamics
of microbial cultures [16]. The reduction of SSL in A.
xylosoxydans TD2 in the medium with TDG promotes
the shortening of both the lag-phase and the duration of
biodegradation process as a whole. The data obtained
allow the calculation of the optimal amount of inoculumculture according to the initial substrate concentration.
This parameter can be used for regulation of the
efficiency of the TDG biodegradation process.
A. xylosoxydans TD2 can grow at rather high TDG
concentrations in the medium, but the maximal values
of specific growth rate and biomass yield were observed
at TDG concentrations not over 10 g/l. No cell
propagation was observed at a concentration of 39.3g/l, however, cells remained metabolically active and
oxidized TDG to TDGA, thus reducing its concentra-
tion in the medium. Growth started when the TDG
Fig. 5. The growth dynamics and metabolates accumulation by A.
xylosoxydans TD2 in the medium with high TDG concentration (j,
OD560; I, TDG; m, DGSO; m, TDGA).
Fig. 4. Accumulation of DGSO and TDGA by A. xylosoxydans TD2
in the medium with TDG in flasks (a) and fermenter (b) (j, OD560; I,
TDG, m, DGSO; m, TDGA).
I.T. Ermakova et al. / Process Biochemistry 38 (2002) 31�/3936
concentration decreased to 25 g/l. Under these condi-
tions the specific growth rate was 0.004 h�1, i.e. several
times lower than that with initial TDG concentration of
26.4 g/l (0.015 h�1).
Evidence of DGSO and TDGA in the medium
suggests that TDG can undergo the microbial attack
through two independent pathways. One of these is the
oxidation of the sulphur atom with the formation of
DGSO. In addition, TDG can be metabolized through
oxidation of primary alcohol groups to TDGA and
subsequent uncoupling of C�/S bonds of TDGA and
TGA.
The uncoupling of this rather stable bond (the energy
of C�/S bond is equal to 64 kcal/mol) [17] is the key
reaction in the degradation of sulphur-containing com-
pounds, since the final products of this process are
readily assimilated by microorganisms in the reactions
of peripheral or central metabolism. Several examples of
such microbial cultures R. rhodochrous , Gordona ai-
chiensis , Brevibacterium sp. , have been reported to split
C�/S bond in dibenzothiophene molecule using it as a
sulphur source. The initial steps of the microbial attack
on dibenzothiophene are the oxidation of the sulphur
atom by dibenzooxygenase with the successive forma-
tion of sulphoxide, sulphone and 2-hydroxy-biphenyl-2-
sulphinate. The uncoupling of the C�/S bond occurs by
means of 2 hydroxy-biphenyl-2-sulphinatelyase with
elimination of a sulphur atom as a sulphate ion and
formation of 2-hydroxy-biphenyl in R. rhodochrous and
G. aichiensis [18�/20] or benzoate in Brevibacterium sp .
[21]. The enzyme complexes of Alcaligenes sp . [22] and
Pseudomonas putida [23], including dioxygenases with a
wide substrate specificity, achieve the uncoupling of the
C�/S bond in aromatic sulphonates.
These results suggest that the oxidation of TDG to
DGSO by A. xylosoxydans TD2 is most probably
catalyzed by an oxygenase with a wide substrate
specificity. However, this enzyme seems to be inactive
with respect to DGSO and sulphoacetic acid containing
a sulphur atom in higher oxidation degrees. A compar-
ison of the results for both A. xylosoxydans ssp.
xylosoxydans (SH91) [10,24] and A. xylosoxydans ssp.
denitrificans TD2 has shown that the transformation of
TDG to DGSO in these cultures is the metabolic
deadlock. Such processes occur during the biodegrada-
tion of xenobiotics and are realized due to a wide
substrate specificity of different enzymes.
The processes of accumulation and utilization of
TDGA by A. xylosoxydans TD2 is largely determined
by cultivation conditions (pH, pO2, TDG concentra-
tion). The acid is accumulated in the medium and not
utilized under unfavourable growth conditions such as
low pH and high substrate concentration. However,
under optimal growth conditions in a fermenter its
accumulation does not occur. The increase in the flow of
this metabolite for following oxidative reactions can be
accounted for in a 4.5-fold decrease in the rate of TDGA
accumulation in the period of active bacterial growth as
compared to the lag-phase (Fig. 5). These results imply
that TDG destruction is realized by the subsequent
transformation reactions of TDGA.
The oxidation of TDG to TDGA can be achieved
with the involvement of alcohol dehydrogenase. Speci-
fically, NAD-dependent butanol dehydrogenase in A.
xylosoxydans ssp. xylosoxydans SH91 was shown to
catalyze the successive formation of S-(2-hydro-
xyethylthio)acetic acid and TDGA from TDG [10].
The sulphur atom in TDG molecule is covalently linked
to two carbon atoms just as it occurs in the methionine
molecule. The uncoupling of the C�/S bond is known to
occur with the involvement of methionine-g-lyase,
resulting in formation of 2-ketobutyrate and
methanthiol, which are successively metabolized in
Table 3
The growth and respiratory activity of A. xylosoxydans TD2 on TDG and its possible oxidation products
Carbon sources Substrate concentration Growth parameters Substrate concentration Respiration activity
g/l mM mmax (h�1) Lag-phase (h) mM Natom O2/mg biomass per min
Thiodiglycol 3.0 24.0 0.04 4 40.0�/160.0 350�/450
Diglycolsulphoxide 0.5�/1.5 3.6�/10.8 No growth 0.7�/3.6 0
Thiodiglycol � 3.0 24.0
Diglycolsulphoxide 0.5�/1.0 3.6�/7.2 0.04 4 n.d. n.d.
Thiodiglycolic acid 0.8 5.3 0.008 72 3.3�/10 120�/172
2.0 13.2 No growth 16 100% inhibition
Thiodiglycol � 3.0 24.0
Thiodiglycolic acid 0.2 1.32 0.04 4 n.d. n.d.
2.0 13.2 0.018 48 n.d. n.d.
Thioglycolic acid 0.5�/1.7 5.5�/18.6 No growth 1.1�/11.0 340�/240
30.0 100% inhibition
Acetic acid 1.0 17.0 0.08 2 10.0�/34.0 150�/20
Sulphoacetic acid 0.5�/1.0 2.7�/5.4 No growth 2.7 0
n.d., no determination was performed.
I.T. Ermakova et al. / Process Biochemistry 38 (2002) 31�/39 37
microbial cells through the dimethylsulphoxide pathway
[25].
The presence of TGA and acetate among the inter-
mediates of TDG and TDGA oxidation suggests similar
mechanism of degradation in A. xylosoxydans TD2.
Thus the results of this work allow to present TDG
degradation pathway in A. xylosoxydans ssp. denitrifi-
cans TD2 as follows (Fig. 6).
According to this scheme, the main catabolic reac-
tions are TDG oxidation to TDGA with subsequent
uncoupling of C�/S bonds in TDGA and TGA resulting
in the formation of acetate. The latter is assimilated in
the reactions of central metabolism providing bacterial
cells with carbon and energy sources. This suggestion is
supported by the fact that sulphate ions as the final
products are produced from TDG in equimolar
amounts.
A. xylosoxydans ssp. denitrificans TD2, which actively
degrades TDG, the product of mustard detoxification,
can be used for bioremediation of soils contaminated by
this CW agent as well as in the technologies for
biological cleaning of the solutions formed during
degassing of containers by the alkaline hydrolysis of
residual mustard.
Acknowledgements
The authors acknowledge to P.B. Terent’ev for the
analysis and interpretation of chromato-mass-spectro-
metric data. This work was supported by grant ‘En-
vironmental Biotechnology’ of the Russian Federal
Scientific and Technical Program ‘Researches and
Developments on Priority Directions of Science and
Civil Engineering’, subprogram ‘The Novel Methods in
Bioengineering’.
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