SOLATION AND CHARACTERIZATION OF PUTATIVE BURKHOLDERIA sp. HY1 FROM MUD THAT ABLE TO DEGRADE 2, 2-DICHLOROPROPIONATE

Abstract

Halogenated organic compound are extensively used as pesticides, herbicides, and antibiotics. However, using these chemicals in agriculture and industry in high concentration will make them extremely harmful to humans and animals. 2, 2-Dichloropropionate (2, 2-DCP) or Dalapon is one example of halogenated organic compounds that used in agriculture that can cause pollution. In this research, a bacterium strain HY1 was isolated from the mud taken from UTM agriculture area. HY1 showed its ability to degrade 2, 2-DCP by observing its growth on 2, 2-DCP liquid minimal media with doubling time of 42.15 hours. Result has shown that this bacterium grew best in 10mM 2, 2-DCP minimal medium. The activity of dehalogenation and growth pattern was directly proportional to the chloride ion released using colorimetric assay with maximum chloride ion released recorded at 0.748mmol/L from growth in 10mM of 2, 2- DCP. The 16S rRNA analysis and biochemical tests were carried out to identify the identity of the bacterium. From 16S rRNA analysis, the bacterium (HY1) had 95% identity to Burkholderia sp. HY1 is Gram negative bacterium and it had many similarities with Burkholderia sp. in terms of microscopic observation and biochemical tests. The PCR technique was carried out to determine dehalogenase gene of Burkholderia sp. whether related to group I or group II according to Hill et al., (1999) classification system. The PCR amplification and sequencing results showed that the primers dhlB-314 and dhlB-637 showed PCR amplification and primers deh H2-1157 and deh H2-1662 (specific to degrade haloacetic acid) did not show any PCR amplification. This result suggests that a bacterium (HY1) only encode group I dehalogenase and its non-steroselectivity is in agreement with group I haloalkonoic acid such as (2,2DCP, D, L, 2-CP). BLASTp results showed that the partial gene had no significant sequence identity to the sequence in the database. It suggests may be it belongs to other group of dehalogenase.