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Background and Objective:
Many pests of agricultural crops are controlled
by the application of herbicides, fungicides and insecticides.
Synthetic chemicals produced for these purposes are often
chlorinated aromatic organic compounds. Chlorinated compounds
usually persist in the environment and may pose health threats to
humans. Pesticides are often biodegraded by bacteria but little is
known of the bacterial biodegradation of “naturally produced”
chlorinated organic compounds and how this relates to the
biodegradation of synthetic chlorinated compounds.
The purpose of this project was to investigate
the bacterial degradation of 3,5-dichloroanisyl alcohol (DCA), a model
naturally produced chlorinated compound similar in chemical structure
to several synthetic pesticides.
Procedure and Project Activities:
Isolation of a Bacterium Capable of Degrading
3,5-Dichloroanisyl Alcohol
A bacterium with the ability to degrade DCA was
isolated from commercially prepared compost. Samples of several
commercial composts were added to sterile bacterial growth medium
lacking a carbon and energy source. DCA was then added to these
flasks as the only source of carbon and energy. The flasks were
agitated on a shaker for 7 days before a sample of the medium was
taken to inoculate a new flask. This was continued for a total of 7
successive transfers until a culture of 3 morphologically different
bacteria was obtained. From this culture a single pure strain was
isolated which was capable of growth with DCA as the carbon source.
Identification of the DCA-Degrading Bacterium
The bacterium was identified by morphological and
biochemical testing. Morphology was determined by Gram-staining.
Biochemical testing was conducted by standard microbiological
techniques and by using the commercial Biolog bacterial identification
system. Further identification was performed by PCR-amplification of
the bacterium=s 16S rRNA gene followed by nucleotide sequencing and
comparison with known bacterial 16S rRNA gene sequences in the
Ribosomal Database (RDB).
Determining the Pathway of Degradation of DCA by the Bacterium
During the degradation of an organic compound,
bacteria often leak intermediates of the degradation pathway into the
medium. The DCA-degrading bacterium was grown in large quantities,
collected and inoculated into a medium containing DCA. Over time
samples were taken from the medium, the cells were removed and the
medium extracted with ethyl acetate to isolate DCA and the
intermediates. The ethyl acetate extracts were reduced in volume and
derivatized prior to analysis by gas-chromatography -
mass-spectroscopy (GC-MS).
Degradation of a Commercial Herbicide
Dichloroanisinic acid is an isomer of the
herbicide Dicamba (Banvel). The DCA-degrading strain of B. gladioli
was inoculated onto medium containing Dicamba as the sole carbon and
energy source to determine if the bacterium was able to degrade
Dicamba.
Results and Discussion:
A bacterium was isolated from commercially
produced compost which could degrade 3,5-dichloroanisyl alcohol.
This bacterium caused the removal of both chloride ions from each
molecule of DCA degraded. The bacterium was identified as
Burkholderia gladioli, a common soil and plant-associated
microorganism. Other species of Burkholderia are well-known for
their abilities to degrade man-made pollutants including
chlorinated organic compounds.
The DCA-degrading Burkholderia gladioli strain
could completely remove low concentrations (< 2 mM of DCA) from the
medium, higher concentrations were toxic. The strain required oxygen
to degrade DCA. GC-MS analysis of the medium as degradation of DCA
was occurring identified several intermediates leading to the proposed
pathway of DCA catabolism.
Protochatchuate is a well-known entry point to
the central metabolic pathways of bacteria in the degradation of many
natural and man-made compounds.
The commercial herbicide Dicamba (Banvel) has a
highly similar structure to DCA. The DCA-degrading strain of B.
gladioli studied in this project was able to also degrade this
herbicide but only at very low concentrations, 0.1 and 0.2 mM. Since
DCA is a naturally produced chlorinated compound and Dicamba is a
man-made, chlorinated compound this work has implications for the
development of biodegradation abilities of soil bacteria for various
pesticides. Soil and especially compost contain bacteria which have a
rudimentary ability to degrade man-made chlorinated compounds such as
pesticides. These bacteria may evolve in the presence of pesticides
to become more efficient pesticide degraders.
There is considerable pressure on agriculture
to reduce or eliminate the use of chlorinated organic compounds as
pesticides. Understanding that there is a natural process leading
to the degradation of chlorinated compounds can help in the removal
of pesticides from the environment. Knowledge gained in this study
indicates bacteria which can naturally degrade chlorinated compounds
and pesticides are present in compost and that composting may lead
to the removal of pesticides from the environment.
Acknowledgements:
The project investigator wishes to thank the
Government of Manitoba and the Government of Canada through the
Agri-Food Research and Development Initiative for funding which made
this research possible. Also, thanks to Manitoba Hydro for their
cash contribution of $22,800 and BASF who kindly provided Dicamba
for part of the project.
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