Characterization of a Labrys sp . strain Wy 1 able to utilize 2 , 2-dichloropropionate ( 2 , 2-DCP ) as sole source of carbon

Industrial chemicals and pesticides from agricultural activities cause a considerable threat to the environment. 2,2-dichloropropionate (2,2-DCP) is a synthetic halogenated compound used as herbicide. High concentration of 2,2-DCP is toxic if released to the environment and may pollute the soil and ground water source. Using current enrichment technique it was expected to isolate a new bacteria species able to degrade -halocarboxylic acid. Strain Wy1 isolated from soil in Melaka rubber estate was able to utilize 30 mM 2,2-DCP as the sole source of carbon and energy with maximum chloride ion released of 0.27 mmol/L in the liquid growth medium. The biochemical test and 16S rRNA analysis suggested that the bacterial identity was from the genus Labrys sp., and therefore it was designated as Labrys sp. strain Wy1. The cells doubling time in 30 mM 2,2-DCP liquid minimal medium was 33.44 h. Cell growth was inhibited when grown in liquid minimal medium above 30 mM 2,2-DCP due to the toxicity of 2,2-DCP to the cells. This is the first reported case that the genus Labrys sp. is able to degrade 2,2-DCP.


INTRODUCTION
Halogenated compounds were used extensively as herbicide and as intermediate chemicals in many industries.Due to the complexity, toxicity, persistence and ubiquitous distribution spreading of these xenobiotic compounds, they have threatened the health and living quality of human and other organisms (Fetzner and Lingens, 1994).Degradation of halogenated compound has been reported since the earlier of 20th century by Penfold (1913).
The enzymes which were responsible for the degradation of halogenated compound were dehalogenases, discovered and named by Jensen (1957).Culturing and enrichment of microorganism that can produce dehalogenase in the presence of halogenated compound in the environment was the most favorable method.Several dehalogenase producing bacteria were isolated using this technique including were Methylobacterium sp.HJ1 (Jing and Huyop, 2008), Pseudomonas putida PP3 (Senior et al., 1976), Xanthobacter autotrophicus GJ10 (Janssen et al., 1985), Pseudomonas B6P (Mesri et al., 2009) and Rhizobium sp.(Berry et al., 1979).Current research also incorporated the methods as described by previous work to increase the probability of isolating reliable bacteria.Therefore, the aim of this study is to isolate and characterize bacteria from Melaka rubber estate agricultural soil which able to utilize 2,2-DCP as only carbon source.Various types of chlorinated pesticides and herbicides were used by the estate owner and we were interested to isolate and characterize the bacteria resistant to chlorinated xenobiotics from the soil.

Sample preparation
Soil sample (5 g) from rubber plantation estate in state of Melaka was mixed together in minimal medium containing 10 mM of 2,2-DCP and incubated in orbital shaker at 30°C.Bacterial growth was monitored at appropriate time interval.For the isolation of pure colonies, 0.1 mL of aliquot was spread onto solid minimal media contained 10 mM 2,2-DCP.For cells purification, colonies were selected and subcultured onto fresh plates containing similar amount of 2,2-DCP as carbon source.Plates were incubated at 30°C.

16S rRNA gene analysis
Genomic DNA from bacterial culture was isolated using Promega® Wizard® Genomic DNA Purification Kit.PCR amplification of the 16S rRNA gene for bacteria identification was carried out using universal primers, FD1 (5'-aga gtttgatcctggctc ag-3') and rP1 (5'acg gtcataccttgttacgac tt-3') as suggested by Weisburg et al. (1991).Each PCR reaction mixture (total volume 50 µL) contained 1 unit/mL Taq polymerase (Promega®), 50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 1.2 mM of MgCl2, 160 µM of each dNTPs, 1µmol of each primer, nuclease free water and 100 ng of DNA template.The PCR protocol consisted of a denaturing process of 94°C for 5 min, followed by 30 cycles of 92°C for 1 min, 55°C for 1 min and 72°C for 2 min, incubation at 72°C for 10 min and finally 4°C for infinite time for storage, using GeneAmp® PCR system 9700.Amplicons were purified using QIAGEN® QIAquick PCR purification kit and sequenced by 1 st BASE® company (Singapore).

Phylogenetic analysis
The 16S rRNA gene sequences were compared with GenBank database using BLAST (Altschul et al., 1997) by selecting BLASTn method.Multiple sequence alignment of bacteria from different genus were constructed using profile alignment command of CLUSTAL-W from MEGA version 4 (Tamura et al., 2007).Using the same software, phylogenetic trees with bootstrap test (1000 replications) were constructed using Neighbour-Joining method (Saitou and Nei, 1987).

Isolation and characterization of novel bacterium able to utilize 2,2-DCP
After 4 days of incubation the colonies appeared to be white, round shape and their size ranging from 0.5-1.0mm on 10 mM 2,2-DCP minimal medium plate.Microscopic observation suggested all purified bacterial colonies were of similar type and further analysis by 16S rRNA genes proved they were all clustered in the same genus and initially designated as strain Wy1.
Growth experiment was carried out (Figure 1).Minimal media supplied with 30 mM 2,2-DCP recorded the highest growth with cells' doubling time 33.44 h (Table 1).However, growth was negligible in 10 and 50 mM of 2,2-DCP.Chloride ion assay showed maximum chloride ion released by bacteria grown in 30 mM 2,2-DCP with approximately 0.27 mmol/L (Figure 2).Biochemical tests were conducted as summarised in Table 2 showed Wy1 generally matches with the most related species of Labrys Neptuniae sp.Liujia-146.

16S rRNA gene analysis and biochemical tests and
A total of 1351 bases of 16S rRNA gene were sequenced.BLASTn showed the isolated bacterium matched to the genus Labrys, where the first 10 species were highly identical (Table 3).The partial sequence of 16S rRNA gene was submitted to Gene Bank with accession number JF907580.All identical bacteria from BLASTn results were used to construct Neighbour-joining phylogenetic tree with bootstrap test.The tree shows bacteria Wy1 was grouped with Labrys neptuniae sp.Liujia-146 with the highest bootstrap value (Figure 3).

DISCUSSION
The main goal of this study is to isolate bacteria which have not been reported to utilize 2,2-DCP as sole carbon and energy source.Generally the bacterial colonies were observed after 4 to 5 days of incubation at 30°C with pH7 in aerobic condition.No colonies were observed in a blank plate contained only minimal media without adding any carbon source.Growth profile showed slow growth of bacterium Wy1 and the maximum absorbance only achieved approximately after 5 days in liquid culture condition (Table 1 and Figure 1).The bacteria grew best in minimal media contain 30 mM 2,2-DCP with the highest absorbance recorded with cells doubling time of 33.44 h.Slow growth of bacteria suggested that the inefficient of the cells uptake system of the growth substrate into the cell (Jing et al., 2010) or weak expression of the dehalogenase gene in the bacteria system.The growth of the isolated bacteria was 3 times slower compared to the previously reported Rhizobium sp. in 2,2-DCP liquid medium (Allison et al., 1982).However, detection of chloride ion released into the  "+" for positive result, "-" for negative result; NR -Not reported; Miller et al. (2005).
growth medium suggested the utilization of 2,2-DCP as a carbon source.
Figure 3 shows the taxonomic position of Wy1 with other closely related species from different family.Wy1 was clustered together with Labrys neptuniae sp.Liujia-146 with high bootstrap value (98), suggesting they were closely related.However, DNA-DNA hybridization test is needed to confirm this finding and define them as same species.According to Chou et al. (2007), Labrys neptuniae sp.Liujia-146 was a budding bacteria found in root nodules of Neptunia oleracea.This is the first reported case of which Labrys sp. could survive solely on 2,2-DCP as carbon source.There has been reported case of Labrys portucalensis sp.nov.which capable of degrading fluorobenzene found in contaminated sediment from northern Portugal (Carvalho et al., 2008).Labrys  (Li et al., 2011).Both bacteria were able to use halogenated compound as sole source of carbon.Current study is the first reported case of the respective bacteria from genus Labrys which could degrade -haloalkanoic acid.
variety of biochemical tests were carried out including oxidase test, catalase test, gelatine liquefaction test, lactose utilization test, Simmons citrate test, indole test, nitrate reduction test, urease test, triple sugar iron (TSI) test and oxidation fermentation (OF) test to

Table 2 .
Comparison of biochemical test with related species.

Table 3 .
Species of bacteria identical to Wy1 16S rRNA gene sequence.