SCIENTIFIC CORRESPONDENCE

A method for screening of bacteria capable of degrading

dimethylformamide
N,N-dimethylformamide (DMF) is an orga-
nic solvent produced in large quantities
throughout the world. It is mainly emplo-
yed in the textile and pharmaceutical in-
dustries as versatile solvent, an additive
and in the synthesis of several organic
compounds1,2. It is estimated that the world-
wide production of DMF is 300,000 tonnes3.
Considerable attention has been devoted
to its possible role in environmental pol-
lution and its toxicity to human beings
and other organisms1. Occupational expo-
sure occurs through inhalation of DMF
vapours and through skin contact. It seems
that in man, the digestive system (liver
and upper gastro-intestinal tract) repre-
sents the main target organ after acute or
chronic industrial exposure4,5. Stomach
pain, abdominal cramps, loss of appetite,
nausea, vomiting, headache, weight loss
and insomnia are the most frequent sub-
jective complaints reported by workers
exposed to 10–30 ppm DMF5–9.
Because of its widespread use in industry,
DMF is commonly found in industrial
effluents. The overall rate of chemical
degradation is expected to be very slow
in surface water10. The photooxidation half-
life of DMF in water was estimated experi-
mentally at 50 days and would be even
longer in the natural environment where
other compounds compete for reactions
with hydroxyl radicals. The rate of hy-
drolysis of amides like DMF at normal
temperature in the laboratory is extremely
slow, even under strong acidic or basic
conditions11,12. The low temperature and
near neutral pH of natural surface water
almost preclude the hydrolysis of DMF
under normal environmental conditions.
Because of its widespread appearance
in industrial effluents, difficulty in its
removal from effluents, toxicity and its
slower rate of degradation, considerable
attention has been given to DMF biodeg-
radation. Although few authors have re-
ported DMF biodegradation1,2,13, none of
them have explained a method for screen-
ing of bacteria capable of degrading
DMF. In view of this, the present investi-
gation was undertaken to design a method
for screening bacteria capable of degrad-
ing DMF and its application in the presence
of additional carbon sources. This report
explains the simple screening method for
DMF-degrading bacteria.
Bacteria capable of degrading DMF
were isolated and tentatively identified as
Pseudomonas sp. and designated as DVK1.
The mineral salts medium (MM1) used
for DMF degradation studies was devoid
of carbon and nitrogen source and its
composition is as follows (g/l): K2HPO4
6.8; KH2PO4 1.2; MgSO4.7H2O 0.1;
MnSO4.4H2O 0.1; CaCl2.2H2O 0.1;
FeSO4.7H2O 0.1; Na2MoO7.2H2O 0.006.
pH of the medium was adjusted to 7.0
and sterilized by autoclaving. The bacterium
was propagated using MM1 medium
supplemented with 0.1 %DMF as sole
source of carbon, nitrogen and energy.
Agar plates were prepared from MM1
medium with 2% agar and the plates
were overlaid with 50 µl DMF.
The shake flask experiment was carried
out with DVK1 in order to confirm DMF
degradation. Four flasks, each containing
50 ml of MM1 medium were autoclaved
and supplemented with 0.4% DMF (v/v).
The flasks were inoculated with seed cul-
ture having 5.5 × 109 CFU/ml, so as to give
an initial optical density of 0.1 at 600 nm.
All the flasks were kept on an orbital
shaker at 30 ± 2°C with 180 rpm for four
days. Next 10 ml of culture broth was
removed from each flask at 24 h interval.
The growth of bacterium was measured
as optical density at 600 nm using a spe-
ctrophotometer. pH of the culture broth
Figure 1. Degradation of DMF by Pseudomnoas sp. DVK1. Bacterium was grown in MM1
medium with 0.4% DMF (v/v) as the sole source of carbon, nitrogen and energy. (˜) Growth of
bacteria; (™) pH of growth medium; (£) mM of ammonia liberated and (¢) Per cent of residual
DMF.
was monitored throughout the experiment,
and then centrifuged at 5000 g for 10 min.
Concentration of liberated ammonia in the
spent medium was measured by Nessler’s
method14. DMF concentration in the
spent medium was estimated by HPLC
analysis.
Results of the growth, utilization of
DMF and production of ammonia by DVK1
are shown in Figure 1. It was observed
that maximum growth of the bacterium is
seen after 60 h and more that 50% of
DMF is degraded within 48 h of incuba-
tion. Further, it is clear that DMF degra-
dation starts by the accumulation of
ammonia in the culture medium. The lib-
erated ammonia is also used by the bac-
terium as a source of nitrogen and excess
ammonia is released to the surrounding
medium. The excess ammonia contributes
to the increase in pH of the growth medium
from an initial value of 7.0 to 9.22. DMF
degradation decreased after 48 h of incu-
bation. This may be due to the fact that
increase in pH of the medium might have
suppressed the growth of the bacterium.
Based on the above results, a simple
screening method for isolation of bacteria
capable of degrading DMF has been deve-
loped. Agar plates were prepared using
MM1 medium containing an indicator
dye, Phenol red (0.02 % w/v) and spread
with 50 µl DMF. Then the plates were

1652 CURRENT SCIENCE, VOL. 87, NO. 12, 25 DECEMBER 2004


Figure 2. pH indicator plate showing degradation of DMF. The plate is prepared as described
in the text. It is divided into three sectors. Sector 1, Control (without bacterium); Sector 2, In-
oculated with bacterium which has no ability to degrade DMF; Sector 3, Pseudomonas sp.
DVK1. Appearance of pink colour in sector 3 indicates DMF degradative ability of DVK1.
Table 1. Degradation of DMF by Pseudomo-
nas sp. DVK1 on indicator plate in the presence
of additional carbon source
Growth of DVK1
on indicator plates
spread with
DMF in addition with
None
Glucose
Acetate
Citrate
Succinate
divided into three sectors: 1, 2 and 3
(Figure 2). Sector 1 is a control where the
bacterium was not inoculated and sector
2 is streaked with a bacterium which is
unable to degrade DMF. Sector 3 is
streaked with DVK1 which has the ability
to degrade DMF. The plates were incubated
at 30 ± 2°C for 4–5 days in an incubator.
The bacterial cultures capable of utiliz-
ing DMF as a source of carbon and nitro-
gen resulted in the release of ammonia.
This released ammonia causes an increase
in pH of the indicator plate, resulting in
the change in colour of the indicator dye
from red to pink (pH 7.0 to 9.22). There
is no change in the colour of indicator
dye in sectors 1 and 2, whereas change in
colour of indicator dye from red to pink
is observed in sector 3. This is because
sector 3 is inoculated with DVK1 which
is capable of degrading DMF with the
CURRENT SCIENCE, VOL. 87, NO. 12, 25 DECEMBER 2004
SCIENTIFIC CORRESPONDENCE
Time taken
to change colour
from red to pink
(in days)
3–4
2–3
2–3
2–3
2
release of ammonia. The liberated ammo-
nia results in the change in colour of the
indicator dye. Based on the above results,
we have screened a number of bacterial
cultures for their ability to degrade DMF
(data not shown). From this it is clear
that one can make use of this technique
to screen a large number of microorganisms
for their ability to degrade DMF within a
short time.
Application of the indicator plate has
also been extended by making use of ad-
ditional carbon sources. It was reported
that the use of secondary carbon sources
enhanced the degradation of xenobiotic
compounds15,16. In this investigation the
indicator plates were prepared with 10 mM
of individual secondary carbon sources
such as glucose, citrate, acetate and succi-
nate. These plates were then spread with
50 µl of DMF and streaked with DVK1.
Bacteria will not utilize these secondary
carbon sources immediately in the MM1
medium. This is because nitrogen is a
limiting factor and has to be supplied by
DMF only after its complete degradation.
Results of DMF degradation with addi-
tional carbon sources by DVK1 are shown
in Table 1. The results indicate that the
plate supplemented with no additional
carbon sources formed pink colour within
3–4 days, whereas indicator plates supplied
with glucose, acetate and citrate formed
pink colour within 2–3 days. The plate
supplemented with succinate formed pink
colour within two days in comparison
with other secondary carbon sources. This
shows that succinate is a more pronounced
secondary carbon source for DMF degra-
dation compared to glucose, acetate and
citrate. This procedure enhances the growth
rate of bacteria and also allows the selection
of strains that have the potential to degrade
DMF.
Liberation of ammonia during degrada-
tion of DMF can be used as an indication
for the activity of bacteria in the growth
medium. With Pseudomonas sp. DVK1,
it could be demonstrated that the liberation
of ammonia is proportional to the degra-
dation of DMF. Under these conditions,
a screening method was developed using
pH indicator dye such as Phenol red, for
detection of DMF-degradative bacteria.
Degradation of xenobiotc compounds some-
times requires secondary carbon sources.
The pH indicator plate allows the selection
of such strains. This method will facilitate
the selection and isolation of DMF-degra-
dative bacteria and allows the study of a
large number of such microorganisms.
This method may be used as a prerequisite
for application in decontamination of such
xenobiotic compounds.
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and Tokuyama, T., J. Ferment. Bioeng.,
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M. R., Clin. Res., 1987, 35, 756A.
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SCIENTIFIC CORRESPONDENCE
7. Scailteur, V. and Lauwerys, R. R., Toxi-
cology, 1987, 43, 31–38.
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Chem. Toxicol., 1986, 9, 147–170.
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10. Hayon, E., Ibata, T., Lichtin, N. N. and
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pedia of Chemical Technology, John
Genetic variation of cotton bollworm, Helicoverpa armigera (Hübner)
of South Indian cotton ecosystem using RAPD markers
Documenting the nature of genetic varia-
tion, magnitude and distribution is necessary
for understanding the behaviour, response
to selection pressure, structure and dyna-
mics of different populations and manage-
ment1–5. Availability of reliable polymor-
phic markers often limits the accurate
estimation of genetic variation among
individuals or different populations. Elu-
cidation of genetic variation in geogra-
phical populations can be an important
aspect to study the pest populations and
their management6. Within an ecosystem,
the extent of genetic variation between
geographical populations depends on
several factors, including gene flow bet-
ween populations, host range and time
since separation7,8. Genetic differences
within and between geographic popula-
tions of an ecosystem are likely to be de-
fined by the population fluxing patterns
as influenced by various ecological factors
in the immediate past and the historical
pressures on the genome9.
Usual DNA-based techniques such as
Restriction Fragment Length Polymor-
phism (RFLP) through Southern hybridiza-
tion and use of microsatellites are expensive;
use of the latter is often hindered by lack
of availability of DNA sequence informa-
tion, though it has inherent advantage10.
Polymerase Chain Reaction (PCR)-based
Random Amplified Polymorphic DNA
(RAPD) approach has been a handy and
convenient alternative technique for in-
vestigations of genetic variation and ge-
nome mapping11,12. Because of the nature
of primer sequences, RAPD analysis
samples the genome more randomly than
1654
Wiley, New York, 1980, vol. 11, 3rd edn,
pp. 263–268.
13. Urakami, T., Kobayashi, H. and Araki,
H., J. Ferment. Bioeng., 1990, 70, 45–47.
14. Vogel, A. L., Quantitative Inorganic
Analysis Including Elementary Instru-
mental Analysis, Low & Bryodne Ltd,
London, 1969, 3rd edn, p. 784.
15. La Pat-Polasko, L. T., McCarty, P. L.
and Zehnder, A. J. B., Appl. Environ.
Microbiol., 1984, 47, 825–830.
16. Stortmann, U. and Roschenthaler, R.,
Curr. Microbiol., 1987, 15, 159–163.
other methods and has been successfully
employed in the construction of linkage
maps13–16. Being simple and non-radio-
active, the technique is quite sensitive and
used to detect genetic variation in many
organisms17–21. It has been extensively
used for molecular fingerprinting22–24, phy-
logenetic analyses25,26, genetic mapping27
and population diversity18,28,29 analysis.
The variation that can be accounted for,
between and within populations through
RAPD, appears to be unlimited. Yet, the
dominance nature of these markers is a
greater leveller and introduces subjectivity
in understanding the structure of popula-
tions, where allelic frequencies of genes
matter. Further, cyclic amplification of
DNA being an extremely powerful techni-
que, RAPD patterns are protocol-sensitive,
which limits the cross comparison of in-
formation generated by this method with
others.
Cotton bollworm, Helicoverpa armigera
(Hübner) is a key pest of cotton and other
crops in India and elsewhere, inflicting
huge crop loss each year. Looking at its
versatility in rapidly evolving resistance
to almost all classes of insecticides and its
ability to thrive on several hosts, there
must be a strong genetic basis governing
the behaviour of H. armigera in making
it a serious pest on several crops. Thus,
the understanding of genetic variation
within and between geographical popula-
tions of H. armigera in the cotton ecosystem
and genome-fluxing patterns, coupled
with estimating resistance folds to each
insecticide can expectedly help in pinning
down the exact causes for such frequent
CURRENT SCIENCE, VOL. 87, NO. 12, 25 DECEMBER 2004
Received 5 June 2004; revised accepted 11
August 2004
Y. VEERANAGOUDA
K. NEELAKANTESHWAR P ATIL
T. B. KAREGOUDAR*
Department of Biochemistry,
Gulbarga University,
Gulbarga 585 106, India
*For correspondence.
e-mail: goudartbk@rediffmail.com
outbreaks and versatility in evolving re-
sistance to insecticides at a faster rate.
Elucidation of gene statements responsible
for insecticide resistance in H. armigera
would bring more light in understanding
the phenomenon and management of the
problem. In the Indian context, a systematic
and concerted effort to view the problem
of insecticide resistance from this pers-
pective is important.
We report in the present study, the genetic
variability as revealed by RAPD in 12 geo-
graphical populations representing the
entire South Indian cotton ecosystem.
Cotton bollworms were collected during
peak incidence from each of the 12 loca-
tions of the South Indian cotton ecosystem:
Nanded, Nagpur and Parbhani (Maharashtra);
Guntur, Madhira and Nalgonda (Andhra
Pradesh); Raichur, Dharwad and Mysore
(Karnataka); Coimbatore, Madurai and
Kovilpatti (Tamil Nadu) (Figure 1). About
20 larvae for each location were randomly
picked for isolation of genomic DNA
separately. The larvae were desensitized
using formalin swab, each larva was dis-
sected and the gut contents were comple-
tely removed to avoid any contamination
of plant DNA. Resulting skin and legs
were used to prepare genomic DNA follow-
ing modified CTAB method. DNA was
further purified by phenol–chloroform
treatment. In order to make a better rep-
resentation of each location, equal amount
of DNA from each of 20 larvae for each
location was pooled and the resulting 12
bulked DNA samples were used for PCR–
RAPD analysis. Bulked DNA was diluted
to 20–40 ng/µl before actually being used