Isolation and characterization of a biodegrading 3-chloropropionic acid Burkholderia cepacia WH 1 isolated from abandoned agricultural land

1Department of Biology, College of Science, Al-Mustansiriyah University, Iraq 2Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Malaysia 3Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Malaysia. 4Iffah Izzati Zakaria, Natural Products and Drug Discovery Center, Malaysian Institute of Pharmaceuticals and Nutraceuticals, National Institutes of Biotechnology Malaysia, Ministry of Science, Technology and Innovation, Block 5-A, Halaman Bukit Gambir, 11700 Pulau Pinang, Malaysia


INTRODUCTION
Halogenated aliphatic compounds are xenobiotic substances produced in large quantities for use in the agricultural and industrial processes (Yan et al., 2010;Abdul Hamid et al., 2011).These substances symbolize an important class of environmental pollutants due to their widespread use as herbicides, fungicides, insecticides and solvents (Fetzner and Lingens, 1994;Bhatt et al., 2007).The utilization of such substances have been linked to several problems such as their persistence and a high tendency to bioaccumulate (Bachas-Daunert et al., 2009;Abel et al., 2012), their being carcinogenic and genotoxic (Jing and Huyop, 2007;Drivon and Ruppin, 1998) and so potentially harmful to human kind and the natural environment.Discovery of alternative bio-based methods that could naturally detoxify these substances would therefore be timely.Bioremediation may be an economical, safe and environmentally friendly (Janssen et al. 2005, Abdul Hamid et al., 2011) means to re-move these xenobiotic substances in comparison to the chemical methods viz.adsorption, ion exchange, liquid-liquid extraction, and chemical oxidation (Arora and Bae, 2014).
Over the past few decades, dehalogenases have become increasingly popular as biocatalysts for the bioremediation of halogenated environmental pollutants.
This may be due to the fact that the enzymes have the natural ability to neutralize the toxic compounds by catalysing the cleavage of carbon-halogen bonds (Thasif et al., 2009).To date, numerous haloalkanoic acid dehalogenases have been isolated from different bacteria and characterised (Hill et al., 1999, Wong andHuyop, 2011).Most pertinently, the haloalkanoic acid dehalogenases have been isolated mainly from soil borne bacteria (Slater et al., 1997); such bacteria may have evolved the ability to metabolize halogenated compounds following continuous exposure.According to literary review, continuous exposure of microorganisms to halogenated compounds is often the driving force for their genetic adaptability to produce dehalogenases that metabolize such compounds (Field and Alvarez, 2004).So far, only few microorganisms have been reported capable of degrading b-halo carboxylic acid (b-HA), including 3-chloropropionic acid (3CP) (Mesri et al. 2009;Yusn and Huyop (2009).Among these dehalogenating microorganisms isolated so far, the Bacillus sp.CGMCC no 4196 has been by far the most impressive.The bacterium was reportedly capable of degrading halogenated compounds as concentrated as 120 mM and 30 mM of 3-chlorobutyrate (Lin et al., 2011).The study described that the degradation of 3CPA by the bacterium was the highest when the resting cells were utilized as the biocatalysts.Therefore, it was suggested that the Bacillus sp.CGMCC no 4196 catalysed the dehalogenation of 3CPA by initially hydrolysing the compound to b-hydroxyl aliphatic acid.This was followed by oxidation of the b-hydroxyl aliphatic acid to b-carbonyl aliphatic acid by the b-hydroxy aliphatic acid dehydroxygenase (Lin et al., 2011).
This present work aimed to isolate and characterize bacteria that could effectively degrade the b-halo carboxylic acid, 3CP.Herein, we report the properties of a new bacteria, designated as WH1, able to utilize 3CP as its sole source of carbon and energy.The bacterium was isolated from agricultural land in the grounds of Universiti Teknologi Malaysia (UTM) which is contaminated with herbicides and pesticides.The identity of the WH1 bacterial isolate was determined using genetic and biochemical methods.Considering the low natural abundance of b-chloroalkanoate-degrading bacteria and the inability of a-chloroalkanoate degrading bacteria to dechlorinate the b-chloro substituted alkanoates, the continuous attempts to isolate b-chlorodegrading bacteria remains crucial.Furthermore, the synergistic interaction between the a-chloro and b-chlorodegrading enzymes may be useful, envisaging their use in bioformulations to effectively treat environments contaminated with both forms of halogenated compounds.

Chemicals.
The various halogenated compounds used in this study were all of analytical grade and purchased from Sigma-Chemical Co. (St.Louis, USA).
Microorganism isolation and identification.The bacterial strain was obtained from agricultural land in UTM that had been extensively exposed to herbicides and pesticides.About 5 g of a soil sample was added into a 250 mL shaker flask containing 100 mL minimal salts medium (pH 7.5) and supplemented with 10 mM 3CP as the sole carbon source.After 10 days of incubation with shaking (200 rpm) at 37°C, an aliquot (0.1 mL) was pour plated onto solidified minimal medium supplemented with 10 mM 3CP.The resulting colonies were selected and screened for 3CP degradation.Among the isolates, a strain that rapidly degraded the b-haloalkanoic acid, 3CP, was chosen for further study.

Determination of bacterial growth.
The amount of bacterial growth was determined using the UV-Vis (Perkin Elmer) method, measuring the absorbance of the growth suspension (A 600nm ) and the release of chloride ions due to degradation of 3CP into the growth medium (A 460nm ).Determination of the free halide ion during the reaction was carried out using the method of Bergman and Sanik (1957) with some modifications.The chloride ion released was defined as 1 mM chloride, equivalent to 1 µmole Cl-/ mL.Next, the cells were harvested by centrifugation (10,000 × g for 10 minutes at 4°C) during the mid-logarithmic phase and the cells were washed with 0.1 M Tris-acetate buffer (pH 7.5) and centrifuged again (10,000 × g for 10 minutes at 4°C).This step was repeated three times before the supernatant was decanted and the cells stored at -20°C until use.

Biochemical tests.
In order to ascertain the identity of the isolate, detection of the morphological properties various biochemical tests were carried out: lactose fermentation (MacConkey's), catalase, oxidase, citrate (Simmons), urease (Christensen's), and lactose, motility and gelatine liquefaction.

BIOLOGTM GEN III MicroPlate identification.
Biolog is a commercial tool used to identify unknown bacteria, consisting of 94 phenotypic tests that include 71 carbon source utilization assays and 23 chemical sensitivity assays.The test panel provides a "Phenotypic Fingerprint" of the microorganism which can then be used to identify isolates down to the species level.The WH1 isolate was first grown on an agar medium and then suspended in a special "gelling" inoculating fluid (IF) using the manufacturer's recommended cell density.Then, the cell suspension was inoculated into the GEN III MicroPlate at 100 µL per well and the microplate was incubated to allow the phenotypic fingerprint to form.Initially, the wells started out colorless, and following incubation the increased respiration in the wells caused the reduction of the tetrazolium redox dye to form a purple color in the positive colonies.In contrast, the negative wells that corresponded to the negative control (without carbon source) remained colorless.A positive control (supplemented with a carbon source) was also used as a reference for the chemical sensitivity assays.After incubation, the phenotypic fingerprints of the purple wells were compared to the Biolog's extensive species library.

DNA extraction.
A DNA purification Kit (Promega Kit) was used to extract the genomic DNA from the WH1 bacterial strain.Following DNA extraction, the concentration of the WH1 bacteria genetic material was approximately 300 ng/µl as measured by Nano-Drop analysis.Next, the extracted DNA was subjected to gel electrophoresis.

PCR amplification of the 16S rRNA, gene sequencing and analysis of the 16S rRNA.
Using universal primers Fd1 and rP1, as suggested by (Weisburg et al., 1991), the 16S rRNA of the WH1 isolate was amplified and the PCR product was visualized by gel electrophoresis.Chromosomal DNA was prepared from the late exponential phase culture using the Wizard Genomic DNA Purification Kit (Promega, USA).The polymerase chain reaction (PCR) was carried out to amplify the 16S rRNA of strain WH1.The universal primers used were Fd1 (5'-AGA GTT TGA TCC TGG CTC AG-3') and rP1 (5'-ACG GTC ACC TTG TTA CGA CTT').The total volume of each amplification reaction was 25 µL which consisted of 12 µl PCR master mix (Fermentas Inc.USA), 1 µl template DNA, 1 µl forward primer (Fd1), 1 µl reverse primer (rP1), and 10 µl nuclease free water.16S rRNAGene amplification was performed for 30 cycles whereby each cycle was set as an initial denaturation phase of 94°C for 5 minutes, followed by denaturation 94°C for 1 minute, annealing 55°C for 1 minute and final extension 72°C for 10 minutes.The PCR product was electrophoresed on an agarose gel (0.8%).For the sequencing reaction, the PCR product was purified using a QIAquick PCR purification kit prior to sequencing (1st Base Laboratory, Selangor).The sequence obtained was compared to other sequences in the public databases using the BLAST search program found in the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov).
Phylogenetic analysis of 16S rRNA gene.The 16S rRNA sequence was aligned and compared with the sequences deposited in the Gene Bank database from the National Center for Biotechnology Information (NCBI), using the BLASTn analysis tool.Next, the generated sequences were selected from the BLASTn and added directly into MEGA6 software.The command of CLUSTAL-W was used to align the first 10 sequences obtained from the BLAST analysis.Subsequently, the analysis of phylogeny and alignment of multiple sequences were conducted using CLUSTAL-W and Phylogeny Construction in MEGA6 software (Saitou and Nei, 1987).

HPLC analysis of 3CP degradation.
Samples of the bacterial growth media were analysed by high performance liquid chromatography (HPLC) to monitor reduction of the 3CP (10 mM) substrate in the reaction media.Samples were passed through a 0.2 µm nitrocellulose filter (Sartorius) to remove bacterial cells and particulates prior to HPLC analysis.The analysis was performed at a flow rate of 1.0 mL/min using isocratic elution with a mobile phase containing aqueous potassium sulphate (20 mM): acetonitrile (60:40).The fractions of the sample were detected with a UV detector equipped with a ZORBAX Eclipse XDB-C18 (4.6 x 150mm, 5µM particle sizes).

Isolation and Characterization of 3CP Degrading Bacteria.
Sampling of the soil obtained from the herbicide-contaminated agricultural land in UTM successfully isolated an indigenous bacterial strain that was designated as strain WH1.The bacterium was found to grow on a minimal media containing 10 mM 3CP as the only carbon source following 6 days of incubation.No colonies were observed on the control plates without 3CP (Figure 1).The basic cellular morphology of the WH1 bacteria showed that the bacteria were generally of circular shape, white (pale brown through sunlight) and formed creamy colored colonies on the 10 mM 3CP minimal agar plates.Gram staining revealed that the WH1 strain was Gram negative, confirmed by formation of post staining pink-red colonies.The morphology and biochemical tests as well as the Biolog™ GEN III Microplate results for the WH1 strain are summarized in Table 1 and  Table 2,

Growth profile and halide ion assay.
The WH1 bacteria exhibited a cell doubling time of 43.62 hours in a 10 mM 3CP media with concomitant release of chloride ion in the growth medium (Figure 2).A maximum chloride ion release rate of 0.194 µmol Cl − /mL was observed for the WH1 strain, and pertinently nearly all of the carbon source (> 99%) in the growth media was consumed after 132 hours of incubation (Figure 3).

Identification
of 3-chloropropionate degrading bacteria using 16S rRNA analysis.The 16S rRNA gene of the WH1 isolate was successfully amplified using the universal primers Fd1 and rP1.Gel electrophoresis of the PCR product showed that the amplified gene matched the predicted size of approximately 1500 bp (Figure 4).Burkholderia cepacia isolation, characterization and biodegradation

Properties
Results Properties Results    Sequencing and analysis of 16S rRNA gene.The PCR product of the WH1 isolate was sequenced by 1st BASE ® Malaysia.A total of 1500 bases for the WH1 gene sequenced were aligned with other species using the BLASTn option.

D-maltose
The results for the BLASTn search and the significant sequenced alignments suggest that the WH1 strain belongs to the genus Burkholderia (Table 3).

Phylogenetic study.
Scientific classification of the isolate WH1 was determined as follows: Kingdom -Bacteria; Phylum -Proteobacteria; Class -Betaproteobacteria; Order -Burkholderiales; Family -Burkholderiaceae; Genus -Burkholderia.Bacterial species of the same families of isolates were selected for the phylogenetic analysis.The 16S rRNA sequence of this bacteria species were obtained from NCBI database and The PCR amplification of 16S rRNA gene on an agarose gel (1%).Lane 1: DNA Ladder (1 kb), Lane 2: Amplification of 16S rRNA gene from bacterium WH1, Lane 3: Negative control (dH 2 O+ Fd1 + rP1 + PCR mix).alignment were made using CLUSTAL W provided by MEGA6.A Neighbour-Joining phylogeny tree with boot strap value was constructed (Figure 5).The results inferred that the bacterium WH1 may be designated as Burkholderia cepacia.
HPLC analysis of 3CP concentration in the bacterial growth medium.Samples of the growth medium were analysed using HPLC at daily intervals to monitor the presence of 3CP.The study revealed that the substrate 3CP was almost fully utilized by the Burkholderia cepacia, consistent with the disappearance of a peak that corresponded to 3CP after 96 hours.In contrast, the concentration of 3CP in the negative control minimal medium (without bacteria) remained constant, inferred from the relatively similar area of the peak at 0 and at 96 hours, hence conclusively indicating the non-utilization of 3CP.The HPLC results for the isolated Burkholderia sp. are summarized in Table 4.

DISCUSSION
The Gram negative WH1 bacterial strain was determined to be a Burkholderia sp.following biochemical and morphological tests.Apart from being Gram-negative, Burkholderia sp. is also a motile, rod-shaped and obligatory aerobic bacterium (Zuleta et al., 2014).The findings of this study are consistent with previous reports of the Burkholderia sp. as one of many bacteria prevailing in the natural environment such as in soils, rhizospheres,

Known concentration
Table 4: Summary of HPLC growth medium analysis for the isolated bacteria strain WH1 using Quantitative HPLC analysis concentration formula.
water, plants, fungi, animals, hospital environments and infected humans (Coenye and Vandamme, 2003;Chiarini, et al., 2006 andVial et al., 2011).Burkholderia has been described to be able to survive for prolonged periods in moist environments.A previous study reported that the Burkholderia sp. is able to adhere on plastic surfaces (including those of medical devices) and produce several enzymes such as elastase and gelatinase (Mahenthiralingam Burkholderia cepacia isolation, characterization and biodegradation et al., 2005), so a relatively low virulence Burkholderia sp. was expected.
The significantly low rate of utilization of 3CP by the Burkholderia sp.(43.62 hours) reflects the low sensitivity of the dehalogenase expression in the bacteria towards the substance (Weightman and Slater, 1987).Review of literature revealed the cell doubling time of the Burkholderia sp. to be comparatively sluggish compared to other previously reported b-halogenated degrading bacteria.A study by Schlömann, et al., (1990) reported that Pseudomonas cepacia was able to grow in media supplemented with 2 mM 4-fluorobenzoate (Schlömann, et al., 1990) and exhibited a cell doubling time of 26-28 hours.Similarly, Pseudomonas sp.B6P clocked an impressive cell doubling time of approximately 7-8 hours in a 20 mM 3CP minimal media (Mesri et al., 2009), one of the fastest cell doubling times ever reported for a Pseudomonas sp. that produces b-dehalogenase.Jing et al. (2008) reported a rather short cell doubling time for the Rhodococcus sp.HJ1 at 10-11 hours grown in a 20 mM 3CP minimal media (Jing et al., 2008).On the contrary, comparison to other a-halogenated degrading microorganisms, Raoutella ornithilolytica afforded a cell doubling time of 23.11 hours in 20 mM 2,2-DCP (Niknam et al. 2014).Interestingly, the Arthrobacter sp.S1 that could utilize a myriad of a-halo compounds such as a-halocarboxylic acid (aHA), 2,2-dichloropropionic acid (2,2-DCP) and D,L-2-chloropropionic acid (D,L-2-CP) as well as b-halocarboxylix acid (bHA) and 3-chloropropionic acid (3CP) as sole carbon source, exhibited cell doubling times of 5 ± 0.2 hours, 7 ± 0.1 hours and 10 ± 0.1 hours, respectively (Bagherbaigi et al., 2013).The variety of observed microbial growth speeds in media supplemented with 3CP may correlate with the well-known toxicity of this halogenated compound (Jing and Huyop, 2007).The sluggish uptake of the compound as the sole carbon source and energy for growth observable in the Burkholderia sp. in this study may be due to the bacteria being more sensitive towards the b-haloacid 3CP (Niknam et al. 2014).
According to Towner and Cockayne (1993), the molecular approach is a common way to find a new genus/species.The 16S rRNA gene sequencing is particularly useful as the 16S rRNA gene is present in all bacteria and is a universal target for bacterial identification that is highly accurate, reliable and reproducible (Kolbert and Persing, 1999;Drancourt et al., 2004).The identification of variations in the nucleotide sequence described would allow for the most precise identification and typing of microorganisms.In this investigation, 16S rRNA gene sequence of the 3CP degrading bacterial strain showed 96% identity to Burkholderia sp.However, analysis of the 16S rRNA gene sequence alone is normally not sufficient to provide conclusive differentiation of strains within a species.Therefore, such deficiency was overcome by the use of BIOLOG®, a molecular tool that complements other species confirmation methods, as a single method of identification can be potentially misleading (Abel et al., 2012).
The results from the Biolog analysis conclusively indicated that the Burkholderia sp. was indeed Burkholderia cepacia.
The ability of the Burkholderia cepacia isolate to degrade 3CP was confirmed by HPLC analysis of the bacterial growth media supplemented with the substance.It is widely accepted and also proven in the literature (Lin et al., 2011) that the product that results from the degradation of 3CP is 3-hydroxypropionic acid (3HP).However, it is impossible to determine the concentrations of 3HP in the growth culture as the liberated 3HP quickly utilized as the growth substrate in the bacterial culture.The consistent decline in the concentration of 3CP in the growth media with the increase in the incubation time strongly suggested that Burkholderia cepacia was capable of degrading and utilizing the substance as a carbon and energy source.Hence, it is suggested that the ability of Burkholderia cepacia to utilize 3CP in this study may be associated with the existence of a cluster of enzymatic reaction or reactions that favoured the dehalogenation of 3CP (Selvamani et al., 2015).The findings in this study also suggest that the b-dehalogenase in Burkholderia cepacia may have an important role in the degradation of b-chlorinated xenobiotics in the environment.
The liberation of chloride ions via the microbe-assisted dehalogenation reaction was monitored by a chloride ion assay (Bergman and Sanik, 1975).The released chloride ions reduced the mercury (III) cyanide to produce mercury (II) chloride, releasing cyanide ions that subsequently interacted with ferric ion (Fe 3+ ) to give the colored ferric thiocyanide complex (Bergman and Sanik, 1957).Based on the slow colorimetric development, it can be said that the Burkholderia cepacia exhibited a rather slow rate of 3CP utilization, attributable to a poor uptake system and/ or low expression of the dehalogenase enzyme (Jing et al., 2010).No growth was observed in the media supplemented with 30 mM 3CP.The outcome suggested that the 3CP was probably toxic to the bacteria at higher concentrations and the presence of excess 3CP could not be tolerated, hence no bacterial growth was observed in this medium.The study therefore proposed that the reaction for the dehalogenation of 3CP by Burkholderia cepacia is by hydrolytic dehalogenation similar to that of the Group II dehalogenase enzyme.This mechanism begins with nucleophilic substitution whereby the hydroxide ion of water attacks the electrophilic carbon atom on the chloride group.This results in the transfer of electron pairs from the electron-rich hydroxide to the electron-poor carbon atom.Since carbon can accommodate only eight electrons in its valence shell, the carbon-chlorine bond subsequently breaks as the carbon-oxygen bond begins to form (van Pée and Unversucht, 2003).The chloride ion is then liberated with the pair of electrons that was once bonded to the carbon atom.
According to literary review, organisms capable of utilizing a halogenated organic compound as their sole carbon and energy source generally have some common characteristics.However, there are some basic steps that must first be satisfied before any classification can be made.First, the organism must either possess or able to synthesize a dehalogenase enzyme capable of removing the substituent halogen(s) in response to the presence of such compound in the environment.Secondly, the dehalogenation product should be non-toxic and easily converted to an intermediate compound in the organism's central metabolic pathway.Thirdly, the halogenated compound should be able to enter the cell either passively or by active transport in order to reach the site of dehalogenase activity.Finally, the halogenated compound should be non-toxic to the organism at normal intracellular concentrations.

CONCLUSION
Discovery of a dehalogenase-producing Burkholderia cepacia makes such bacteria very unique since there are no reports to date of such a species able to degrade 3CP as a sole carbon source.Therefore, Burkholderia cepacia is undoubtedly an uncommon source of 3CP dehalogenase enzyme in nature and the dehalogenation properties and capabilities of the bacteria deserve to be assessed and further optimized.The b-halogenated degrading class of dehalogenases is highly valuable in the bioremediation of environments contaminated with the recalcitrant b-halogenated xenobiotic compounds.In view of the current preferred use of chemicals for weed management in the agricultural sector, an increase in utilization and contamination of ecosystems with halogenated compounds are therefore expected.The problem is further exacerbated with the ever growing human population, resulting in the expansion of agricultural land area used for planting crops to meet the increasing food demand.Therefore, the feasibility of utilising aand b-dehalogenases to complement the current treatments in detoxifying halogenated compounds in the environment merits special consideration.Such enzymes may be useful in the bioremediation of environments contaminated with these classes of organohalogens. respectively.

Figure 1 .
Figure 1.Growth of the dehalogenase producing bacteria WH1 on spread plate minimal media containing 10 mM 3CP following 6 days of incubation at 37 o C.

Figure 2 .
Figure 2. The growth profile for the bacterial strain WH1 in minimal media at concentrations of 10 mM, 20 mM and 30 mM 3CP, respectively.

Figure 3 .
Figure 3. Correlation between the growth of bacteria WH1 and rate of chloride ion release in media supplemented with 10 mM 3CP.

Figure 5 .
Figure 5. Neighbour-Joining phylogeny tree of the bacterial isolate WH1.The accession number of each species of bacteria is also provided.

Table 1 .
Morphological and biochemical characteristics of the isolated bacterium WH1 grown on 10 mM of 3CP minimal media.

Table 2 .
Extensive biochemical analysis of the bacterial strain WH1 using BIOLOG™ GEN III Microplate.

Table 3 .
The BLASTn results according to NCBI database.