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A method for screening of bacteria capable of degrading

N,N-dimethylformamide (DMF) is an orga- Bacteria capable of degrading DMF was monitored throughout the experiment,
nic solvent produced in large quantities were isolated and tentatively identified as and then centrifuged at 5000 g for 10 min.
throughout the world. It is mainly emplo- Pseudomonas sp. and designated as DVK1. Concentration of liberated ammonia in the
yed in the textile and pharmaceutical in- The mineral salts medium (MM1) used spent medium was measured by Nessler’s
dustries as versatile solvent, an additive for DMF degradation studies was devoid method14. DMF concentration in the
and in the synthesis of several organic of carbon and nitrogen source and its spent medium was estimated by HPLC
compounds1,2. It is estimated that the world- composition is as follows (g/l): K2HPO4 analysis.
wide production of DMF is 300,000 tonnes3. 6.8; KH2PO4 1.2; MgSO4.7H2O 0.1; Results of the growth, utilization of
Considerable attention has been devoted MnSO4.4H2O 0.1; CaCl2.2H2O 0.1; DMF and production of ammonia by DVK1
to its possible role in environmental pol- FeSO4.7H2O 0.1; Na2MoO7.2H2O 0.006. are shown in Figure 1. It was observed
lution and its toxicity to human beings pH of the medium was adjusted to 7.0 that maximum growth of the bacterium is
and other organisms1. Occupational expo- and sterilized by autoclaving. The bacterium seen after 60 h and more that 50% of
sure occurs through inhalation of DMF was propagated using MM1 medium DMF is degraded within 48 h of incuba-
vapours and through skin contact. It seems supplemented with 0.1 %DMF as sole tion. Further, it is clear that DMF degra-
that in man, the digestive system (liver source of carbon, nitrogen and energy. dation starts by the accumulation of
and upper gastro-intestinal tract) repre- Agar plates were prepared from MM1 ammonia in the culture medium. The lib-
sents the main target organ after acute or medium with 2% agar and the plates erated ammonia is also used by the bac-
chronic industrial exposure4,5. Stomach were overlaid with 50 µl DMF. terium as a source of nitrogen and excess
pain, abdominal cramps, loss of appetite, The shake flask experiment was carried ammonia is released to the surrounding
nausea, vomiting, headache, weight loss out with DVK1 in order to confirm DMF medium. The excess ammonia contributes
and insomnia are the most frequent sub- degradation. Four flasks, each containing to the increase in pH of the growth medium
jective complaints reported by workers 50 ml of MM1 medium were autoclaved from an initial value of 7.0 to 9.22. DMF
exposed to 10–30 ppm DMF5–9. and supplemented with 0.4% DMF (v/v). degradation decreased after 48 h of incu-
Because of its widespread use in industry, The flasks were inoculated with seed cul- bation. This may be due to the fact that
DMF is commonly found in industrial ture having 5.5 × 109 CFU/ml, so as to give increase in pH of the medium might have
effluents. The overall rate of chemical an initial optical density of 0.1 at 600 nm. suppressed the growth of the bacterium.
degradation is expected to be very slow All the flasks were kept on an orbital Based on the above results, a simple
in surface water10. The photooxidation half- shaker at 30 ± 2°C with 180 rpm for four screening method for isolation of bacteria
life of DMF in water was estimated experi- days. Next 10 ml of culture broth was capable of degrading DMF has been deve-
mentally at 50 days and would be even removed from each flask at 24 h interval. loped. Agar plates were prepared using
longer in the natural environment where The growth of bacterium was measured MM1 medium containing an indicator
other compounds compete for reactions as optical density at 600 nm using a spe- dye, Phenol red (0.02 % w/v) and spread
with hydroxyl radicals. The rate of hy- ctrophotometer. pH of the culture broth with 50 µl DMF. Then the plates were
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 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
explains the simple screening method for bacteria; (™) pH of growth medium; (£) mM of ammonia liberated and (¢) Per cent of residual
DMF-degrading bacteria. DMF.

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

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-
Figure 2. pH indicator plate showing degradation of DMF. The plate is prepared as described dation compared to glucose, acetate and
in the text. It is divided into three sectors. Sector 1, Control (without bacterium); Sector 2, In- citrate. This procedure enhances the growth
oculated with bacterium which has no ability to degrade DMF; Sector 3, Pseudomonas sp. rate of bacteria and also allows the selection
DVK1. Appearance of pink colour in sector 3 indicates DMF degradative ability of DVK1. of strains that have the potential to degrade
Liberation of ammonia during degrada-
Table 1. Degradation of DMF by Pseudomo- tion of DMF can be used as an indication
nas sp. DVK1 on indicator plate in the presence for the activity of bacteria in the growth
of additional carbon source medium. With Pseudomonas sp. DVK1,
it could be demonstrated that the liberation
Growth of DVK1 Time taken
on indicator plates to change colour of ammonia is proportional to the degra-
spread with from red to pink dation of DMF. Under these conditions,
DMF in addition with (in days) a screening method was developed using
pH indicator dye such as Phenol red, for
None 3–4 detection of DMF-degradative bacteria.
Glucose 2–3 Degradation of xenobiotc compounds some-
Acetate 2–3 times requires secondary carbon sources.
Citrate 2–3
The pH indicator plate allows the selection
Succinate 2
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.
divided into three sectors: 1, 2 and 3 release of ammonia. The liberated ammo-
This method may be used as a prerequisite
(Figure 2). Sector 1 is a control where the nia results in the change in colour of the
for application in decontamination of such
bacterium was not inoculated and sector indicator dye. Based on the above results,
xenobiotic compounds.
2 is streaked with a bacterium which is we have screened a number of bacterial
unable to degrade DMF. Sector 3 is cultures for their ability to degrade DMF
streaked with DVK1 which has the ability (data not shown). From this it is clear
to degrade DMF. The plates were incubated that one can make use of this technique 1. Yoshie, H., Masaki, M., Yoshinori, S.
at 30 ± 2°C for 4–5 days in an incubator. to screen a large number of microorganisms and Tokuyama, T., J. Ferment. Bioeng.,
The bacterial cultures capable of utiliz- for their ability to degrade DMF within a 1997, 84, 543–547.
ing DMF as a source of carbon and nitro- short time. 2. Bromely-Challenor, K. C. A., Caggino,
gen resulted in the release of ammonia. Application of the indicator plate has N. and Knapp, J. S., J. Ind. Microbiol.
This released ammonia causes an increase also been extended by making use of ad- Biotechnol., 2000, 25, 8–16.
in pH of the indicator plate, resulting in ditional carbon sources. It was reported 3. Marsella, J. A., In Kirk–Othmer Encylo-
the change in colour of the indicator dye that the use of secondary carbon sources pedia of Chemical Technology (eds Kirk,
R. E. et al.,), Wiley, New York, vol. 11,
from red to pink (pH 7.0 to 9.22). There enhanced the degradation of xenobiotic
4th edn, 1994, pp. 967–976.
is no change in the colour of indicator compounds15,16. In this investigation the
4. Potter, H. P., Arch. Environ. Health,
dye in sectors 1 and 2, whereas change in indicator plates were prepared with 10 mM 1973, 27, 340–341.
colour of indicator dye from red to pink of individual secondary carbon sources 5. Lauwerys, R. R. et al., Int. Arch. Occup.
is observed in sector 3. This is because such as glucose, citrate, acetate and succi- Environ. Health, 1980, 45, 189–203.
sector 3 is inoculated with DVK1 which nate. These plates were then spread with 6. Redlich, C. A., Beckett, W. S. and Cullen,
is capable of degrading DMF with the 50 µl of DMF and streaked with DVK1. M. R., Clin. Res., 1987, 35, 756A.

CURRENT SCIENCE, VOL. 87, NO. 12, 25 DECEMBER 2004 1653

7. Scailteur, V. and Lauwerys, R. R., Toxi- Wiley, New York, 1980, vol. 11, 3rd edn, Received 5 June 2004; revised accepted 11
cology, 1987, 43, 31–38. pp. 263–268. August 2004
8. Kinnedy, G. L. and Sherman, H., Drug 13. Urakami, T., Kobayashi, H. and Araki,
Chem. Toxicol., 1986, 9, 147–170. H., J. Ferment. Bioeng., 1990, 70, 45–47. Y. VEERANAGOUDA
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Arbeitsch. Prophyl., 1967, 17, 206–208. Analysis Including Elementary Instru-
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Simic, M., J. Am. Chem. Soc., 1970, 92, London, 1969, 3rd edn, p. 784.
3898–3903. 15. La Pat-Polasko, L. T., McCarty, P. L. Department of Biochemistry,
11. Fersht, A. R. and Requena, Y., J. Am. and Zehnder, A. J. B., Appl. Environ. Gulbarga University,
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Genetic variation of cotton bollworm, Helicoverpa armigera (Hübner)

of South Indian cotton ecosystem using RAPD markers
Documenting the nature of genetic varia- other methods and has been successfully outbreaks and versatility in evolving re-
tion, magnitude and distribution is necessary employed in the construction of linkage sistance to insecticides at a faster rate.
for understanding the behaviour, response maps13–16. Being simple and non-radio- Elucidation of gene statements responsible
to selection pressure, structure and dyna- active, the technique is quite sensitive and for insecticide resistance in H. armigera
mics of different populations and manage- used to detect genetic variation in many would bring more light in understanding
ment1–5. Availability of reliable polymor- organisms17–21. It has been extensively the phenomenon and management of the
phic markers often limits the accurate used for molecular fingerprinting22–24, phy- problem. In the Indian context, a systematic
estimation of genetic variation among logenetic analyses25,26, genetic mapping27 and concerted effort to view the problem
individuals or different populations. Elu- and population diversity18,28,29 analysis. of insecticide resistance from this pers-
cidation of genetic variation in geogra- The variation that can be accounted for, pective is important.
phical populations can be an important between and within populations through We report in the present study, the genetic
aspect to study the pest populations and RAPD, appears to be unlimited. Yet, the variability as revealed by RAPD in 12 geo-
their management6. Within an ecosystem, dominance nature of these markers is a graphical populations representing the
the extent of genetic variation between greater leveller and introduces subjectivity entire South Indian cotton ecosystem.
geographical populations depends on in understanding the structure of popula- Cotton bollworms were collected during
several factors, including gene flow bet- tions, where allelic frequencies of genes peak incidence from each of the 12 loca-
ween populations, host range and time matter. Further, cyclic amplification of tions of the South Indian cotton ecosystem:
since separation7,8. Genetic differences DNA being an extremely powerful techni- Nanded, Nagpur and Parbhani (Maharashtra);
within and between geographic popula- que, RAPD patterns are protocol-sensitive, Guntur, Madhira and Nalgonda (Andhra
tions of an ecosystem are likely to be de- which limits the cross comparison of in- Pradesh); Raichur, Dharwad and Mysore
fined by the population fluxing patterns formation generated by this method with (Karnataka); Coimbatore, Madurai and
as influenced by various ecological factors others. Kovilpatti (Tamil Nadu) (Figure 1). About
in the immediate past and the historical Cotton bollworm, Helicoverpa armigera 20 larvae for each location were randomly
pressures on the genome9. (Hübner) is a key pest of cotton and other picked for isolation of genomic DNA
Usual DNA-based techniques such as crops in India and elsewhere, inflicting separately. The larvae were desensitized
Restriction Fragment Length Polymor- huge crop loss each year. Looking at its using formalin swab, each larva was dis-
phism (RFLP) through Southern hybridiza- versatility in rapidly evolving resistance sected and the gut contents were comple-
tion and use of microsatellites are expensive; to almost all classes of insecticides and its tely removed to avoid any contamination
use of the latter is often hindered by lack ability to thrive on several hosts, there of plant DNA. Resulting skin and legs
of availability of DNA sequence informa- must be a strong genetic basis governing were used to prepare genomic DNA follow-
tion, though it has inherent advantage10. the behaviour of H. armigera in making ing modified CTAB method. DNA was
Polymerase Chain Reaction (PCR)-based it a serious pest on several crops. Thus, further purified by phenol–chloroform
Random Amplified Polymorphic DNA the understanding of genetic variation treatment. In order to make a better rep-
(RAPD) approach has been a handy and within and between geographical popula- resentation of each location, equal amount
convenient alternative technique for in- tions of H. armigera in the cotton ecosystem of DNA from each of 20 larvae for each
vestigations of genetic variation and ge- and genome-fluxing patterns, coupled location was pooled and the resulting 12
nome mapping11,12. Because of the nature with estimating resistance folds to each bulked DNA samples were used for PCR–
of primer sequences, RAPD analysis insecticide can expectedly help in pinning RAPD analysis. Bulked DNA was diluted
samples the genome more randomly than down the exact causes for such frequent to 20–40 ng/µl before actually being used

1654 CURRENT SCIENCE, VOL. 87, NO. 12, 25 DECEMBER 2004