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: starovoitov@ibpm.serpukhov.su (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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