BIODEGRADATION OF MONOCHLOROACETIC ACID BY

IMMOBILIZATION OF XANTHOBACTER AUTOTROPHICUS GJ10 IN

POLYACRYLAMIDE GEL

E. Vasileva, K. Petrov and V. Beschkov

Institute of Chemical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria
Correspondence to: Kaloyan Petrov
E-mail: kaloian04@yahoo.com

ABSTRACT
The degradation of monochloroacetic acid (MCA) by Xanthobacter autotrophicus GJ10 has been studied. The following
processes have been explored: the growth kinetics, the MCA degradation, the glycolic acid production and its further
conversion into chlorides. Immobilization experiments have been conducted with microbial cells entrapped in polyacrylamide
gels. Different ratio of the monomer – acrylamide (AA) and the cross-linking agent N,N’-methylene-bis-acrylamide (MBAA)
was immobilization examined, as the above constituents were varied from 5% to 15% (w/v) and from 0.5% to 1.5%,
respectively. The gels were used in a repeated-batch process, which were compared with the free culture degradation kinetics,
as the quantity of the immobilized cells was 40 times higher than that of the free culture at analogous conditions and
substrates.
Best results were achieved with polyacrylamide gel containing: 10% (w/v) concentration of the monomer AA, 0.1% (w/v) of the
cross-linking agent MBAA, 0.67 ml 5% ammonium persulfate (NH4)2S2O8 and 0.5 ml 2.5% N,N,N′,N′-tetramethylethylene
diamine (TEMED) for 10 ml gel. That culture degrades completely MCA with 10 mM starting concentration. The kinetics of
gel’s exhaustion was investigated in repetitive runs.

Keywords: immobilization, monochloroacetic acid, strain in polyacrylamide gel, aiming to increase the process
polyacrylamide of MCA degradation.

Introduction Materials and methods

Many studies have demonstrated that MCA is the most toxic
halo acetate to aquatic life (5). On the other hand chloroacetic
acid has relatively long lifetime in natural environment (2).
Therefore, much scientific interest focused on biodegradation
of MCA as a potential remedy. Many bacterial strains
possessing inducible dehalogenases are capable of cleaving
off carbon-halogen bond in chloroacetate (3).
Janssen et al. (4) reported that Xanthobacter
autotrophicus GJ10 strain is able of complete mineralization
of 1,2-dichloroethane via metabolic pathway involving halo
acetate dehalogenase activity toward MCA. Biodegradation
of MCA by the strain is confirmed by M. Torz and V.
Beschkov (7), investigating the influence of different carbon
substrates of inoculum in batch and continuous processes.
In the present paper we have studied the MCA utilization
by immobilized cells of Xanthobacter autotrophicus GJ10
Bacterial strain, media and cultivation conditions
X. autotrophicus GJ10 was obtained from the Department of
Biochemistry, University of Groningen, (The Netherlands)
through the National Bank for Industrial Microorganisms and
Cell Cultures, Bulgaria (NBIMCC).
The MMY medium contained (per liter): 5.37g of
Na2HPO4 .12H2O, 1.36 g of KH2PO4, 0.5g of (NH4)2SO4, 0.2
g of MgSO4.7H2O, and 0.015 g of CaCl2.2H2O,
supplemented with 1 ml of trace element solution (7) per liter
and adjusted to pH 7.2 before being autoclaved. The vitamin
solution was substituted by addition of 0.25 g/l yeast extract.
The culture was grown in 500-ml glass flasks, which
contained 200 ml of medium.
The experiments with free and immobilized cells were
carried out in shaking flasks at 30oC on a rotary shaker at 90
rpm. The runs with free and immobilized culture were carried

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out at initial concentration of MCA of 5, 10, 15 and 20mM.
Immobilization procedure
Cell entrapment was performed by the method of Petrov et al.
(6). The active culture for inoculation was prepared in flasks
at 37ºC for 24 h by use of a rotary shaker (New Brunswick
Scientific, USA) at 90 rpm. The cells for immobilization had
been washed twice with 0.9% NaCl and centrifuged at 5000
rev/min for 30 min.
The gel contained: monomer: acrylamide (AA); cross-
linking agent: N,N’-methylene-bis- acrylamide (MBAA);
initiator: ammonium persulfate (NH4)2S2O8; co-initiator:
N,N,N’,N’ tetramethylethylene diamine (TEMED) (all
Sigma).
Gels with different concentrations of AA (5-15% (w/v)),
MBAA (0.1-1% (w/v)), and different MBAA/AA ratio have
been prepared. Each gel contained further: ammonium
persulfate (10 ml 5% w/v solution for 100 ml gel), TEMED
(10 ml 2.5% vol. solution for 100 ml gel) and previously
prepared cell suspension.
The gels were chopped into particles with an average size
of 3 mm of a polyhedric shape. The gel particles were
washed with sterile distilled water and immersed in a 0.9%
NaCl solution after each run, in order to avoid immediate
contamination of the fresh substrate solution at the start of the
next run. When the particles were not used, they were
immersed in 0.9% NaCl and stored in a refrigerator at 4°C.
Analytical methods
The growth of the biomass has been monitored by the optical
density of the broth (OD550) using a spectrophotometer
(uv/vis Spectrophotometer HEλIOS β, Unicom). Dry cell
concentrations were calculated from the optical density, using
a calibration curve.
The quantification of monochloroacetic and glycolic acids
concentrations was carried out by a high-performance liquid
chromatography (HPLC) system, using Bio-Rad column for
organic acids analysis (Aminex Ion Exclusion HPX-87H) and
Knauer variable wavelength detector at 210 nm. As a mobile
phase a 0.01 N sulphuric acid was used at an elution flow rate
of 0.6 ml/min. All samples tested were preliminary purified
through a membrane filter (0.45 μm pores).
Chlorides were determined by a spectrophotometric
method (1). A sample of centrifuged incubation mixture (2.5
ml) was mixed with 1 ml of Fe (III) solution (8 g
(NH4)Fe(SO4).12H2O in 100 ml of 6M HNO3) and 3 ml
saturated solution of 1.5g Hg(SCN)2 in 500 ml of 98%
ethanol. The mixture was shaken and after 10 min filtered
BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE 789
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through 0.2 μm filter. The adsorption was measured at
wavelength 460 nm.
Results and Discussion
Fermentation by free cells
First, we have studied the process in a free culture at different
initial MCA concentrations. Some of these results are shown
in Fig. 1. It is evident that the conversion of MCA to glycolic
acid (GLA) takes place mainly in the phase of exponential
growth. It is seen moreover, that at initial MCA concentration
over 5mM, was observed strong substrate inhibition.
Practically it is impossible to convert initial concentration
more than 10 mM MCA.
10
Concentration [mM]
8 MCA
GLA
6 Chloride
4 MCA
GLA
2 Chloride
0
0 50 100 150 200
Time [h]
Fig. 1. Production kinetics of GLA and chloride, obtained by free culture
from 5 mM MCA (opened symbols) and 10 mM MCA (closed symbols).
Fermentation by immobilized cells
A number of polyacrylamide gels, containing immobilized
Xanthobacter autotrophicus GJ10 cells were examined. A
concentration of the monomer (AA) was varied from 5% to
15% (w/v), the cross-linking agent (MBAA) - from 0.5% to
1.5% (w/v). Best results were achieved with polyacrylamide
gel containing: 10% (w/v) concentration of the monomer AA,
0.1% (w/v) of the cross-linking agent MBAA, 0.67 ml 5%
ammonium persulfate (NH4)2S2O8 and 0.5 ml 2.5%
N,N,N′,N′-tetramethylethylene diamine (TEMED) for 10 ml
gel. That culture degrades completely MCA with 10 mM
starting concentration. The degradation kinetics of MCA,
GLA and chlorides accumulation (Fig. 2) showed very long
lag-phase, due to increased inhibition effect by the synergic
action of MCA and the toxic gel’s ingredients (non-
polymerized acrylamide residua).
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 10 10

Chloroacetic acid [mM]

Concentrtions [mM]

8 8 Run1
Run2
6 MCA 6 Run3
GLA
4 4 Run4
Chloride
Run5
2 2 Run6
0 0
0 100 200 300 400 500 0 100 200 300 400 500
Time [h] Time [h]

Fig. 2. Kinetics of MCA degradation, GLA production and chloride Fig. 4. Degradation kinetics of MCA by immobilized culture of
accumulation by immobilized culture of Xanthobacter autotrophicus GJ10 in Xanthobacter autotrophicus GJ10 during six consecutive batches with initial
batch process with initial concentration 10 mM MCA. concentration of 10 mM MCA.

With the following runs, as a result of microbial growth

Conclusions
inside the particles, the cell leakage rate increases and the Cells of Xanthobacter autotrophicus GJ10 were successively
kinetic curves become more and more similar and closer to immobilized in polyacrylamide gel, containing 10% AA
the curve for free culture fermentation, resulting in the (w/v) and 0.1% MBAA (w/v). When the media contain
shortening of the lag-phase (Fig. 3, 4). 10mM initial concentration of MCA, the immobilized culture
could be used repetitively up to six times in batch processes,
0.35 before the gel’s exhaustion. Assumed the immobilized
0.3 Free Culture
culture degraded over than 50mM MCA in six repeated batch
0.25 Run1
processes. All processes continued until the concentrations of
Dry Cells [g/l]

Run2
0.2 MCA dropped below the limit of detection. When a higher
Run3
0.15 concentration of MCA was used, an increased inhibition
Run4
0.1 effect by the synergic action of MCA and the toxic gel’s
Run5
ingredients didn’t allow MCA consumption.
0.05 Run6
0
0 100 200 300 400 500
Time [h] REFERENCES
1. Bergmann J. and Sanik J. (1957) Anal. Chemistry, 29,
Fig. 3. Comparative time profiles of biomass formation by free and 241-243.
immobilized culture of Xanthobacter autotrophicus GJ10 during six 2. Ellis D., Hanson M., Sibley P., Shadid T., Fineberg
consecutive batches with initial concentration of 10 mM MCA.
N., Solomon K., Muir D. and Mabury S (2001)
Chemosphere, 42, 309-318.
In all cases MCA degradation was accomplished mainly
3. Hardman D. and Slater J. (1981) J. Gen. Microbiol.,
by the free cells, whereas the gel particles served as a donor
123, 117-128.
of cells. During the last run, however, the lag-phase became
4. Janssen D., Scheper A., Dijkhuizen L. and Witholt B.
longer and similar to that of the first run. It means that the
(1985) Appl. Environ. Microbiol., 49, 673-677.
leakage of cells from the particles during the previous runs
5. Kuhn R. and Pattard M. (1990) Water Res., 24, 31-38.
has led to exhaustion and to immobilized cell concentrations
6. Petrov K., Petrova P. and Beschkov V. (2007) World
decrease.
J. Microbiol. Biotechnol., 23, 423-428.
7. Torz M. and Beschkov V. (2005) Biodegradation, 16,
423-433.

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