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ABSTRACT

1,2-Dichloroethane (1,2-DCA) also known as ethylene dichloride is the major


constituents for the production of vinyl chloride. Leaking of 1,2-DCA may contaminate the
underground water source and these chemicals may trigger the onset of cancer in concumer.
Bioremediation is one of the alternative for treatment of contaminated site. Bioremediation
involve the usage of biological agents such as microorganisms in turning hazardous substance to
less hazardous or non-hazardous. A number of microorganisms have been reported to be able to
degrade chlorinated hydrocarbon into products that are harmless to other organisms and the
environment. The growth of these organisms and the rate of degradation can be further enhanced
by optimizing the growth factors such as temperature, dissolved oxygen concentration and
addition of nutrients.

A leakage of 1,2-DCA in Petronas Vinyl Chloride Sdn. Bhd. plant in Kerteh, Terengganu
have cause a major concern to the surrounding community. A study have been conducted to
optimize the biodegradation of 1,2-DCA using in situ microorganisms. The objectives of this
study are to screen for potential bacteria that are capable of degrading 1,2-DCA, to identify the
species of the biodegrading EDC bacteria and finally to identify the optimum physical and
nutrient factor that enhance the growth of the biodegrading bacteria.

Samples were taken from the site of contamination for screening of potential 1,2-DCA
degrading bacteria. Two different well with three different depth for each well were used as
sampling source (depth at 5 m, 10 m, and 15 m). Single colonies bacteria that are isolated are
further purified and screen with different concentration of 1,2-DCA in minimal salt medium
(MSM) agar. The bacteria is then identified by sequencing of the 16S ribosomal subunit using
PCR. The concentration of 1,2-DCA that were used are at 1000 ppm, 2000 ppm and 4000 ppm.
The potential bacteria are then tested with different physical parameters of temperature and
rotation by adjusting the rotary shaker and the pH by adjusting the pH of the medium during
preparation. Nutrients of molas and glucose are also tested to choose the best carbon source and
added to the medium. The best nitrogen source between ammonium chloride and ammonium
sulfate is also determined. The best concentration of both carbon and nitrogen are also determine
with further testing. The best bacteria is chosen and incubated for 10 days with the oprimized
growth parameters and the remaining 1,2-DCA is determined using GC-MS.

Early isolation give a total of 68 isolates successfully obtained from the contaminated site
and only a total of 11 isolates show the potential to degrade EDC as it is able to grow on top of
1,2-DCA up to 4000 ppm in concentration containing in MSM. Among the isolates, S2P5 have
shown the best degradation of 1,2-DCA and have been identified as Bacillus subtilis. Further
testing have shown that the isolates grow best at 150 rpm shaker rotation, temperature at 35C
and pH at 6.5. Addition of molas as additional carbon source have shown an increase in growth
of bacteria, however, increasing the level of nitrogen does not give any positive response. 10
days incubation are collected and placed into a gas chromatography and additional of molas at
2% show the best degradation at 50.85% and releasing -Terpineol and chloroxylenol as the
main products.

The optimum temperature recorded is at 35C which is around the average distribution
temperature in Malaysia. The boiling point for 1,2-DCA is at 83C, therefore the temperature
used for testing will not affect the result obtained. Previous findings have shown that the
temperature for degradation of similar compound is between 25-35C. The optimum rotation at
150 rpm indicate the more oxygen dissolve into the medium. Increase in the number of dissolved
oxygen lead to increase in the metabolic activities of the microorganisms. pH at 6.5 is best suited
for B. subtilis which indicate that the bacterium work best at neutral pH and the usage of neutral
pH will not alter the chemical composition of the 1,2-DCA. Molas usage have been reported in
previous studies and it have shown to be able to increase the degradation rate better than most
carbon-containing supplementation. This is due to the presence of disaccharide chain that are
able to supply a huge number of carbon for the growth of bacteria. The addition or
supplementation of nitrogen source on the other hand does not show any significant increase in
the number of bacteria. This is also supported by previous findings that have reported little to no
changes in the biodegrading rate of 1,2-DCA.