Isolation, biochemical and molecular characterization of 2-chlorophenol-degrading Bacillus isolates

Pure cultures of 2-chlorophenol degrading bacteria were isolated from a natural enrichment that may be adapted to chlorophenols in the industrial zone at Umm-Saied city (Qatar). The bacteria were identified by 16S rDNA analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (~ 550 bp) in the GenBank database revealed that these bacteria are related to the genus Bacillus. Molecular heterogeneity among 2-chlorophenol-degrading bacteria was investigated using REP-PCR chromosomal fingerprinting and correlated with antibiotic profile analysis. REP-PCR results strongly confirmed that the bacterial isolates from different Qatari soils produced different fingerprinting patterns. The distribution of phenol hydroxylase catabolic gene among examined isolates revealed that three isolates out of six yielded positive PCR products. Degradation of 2-chlorophenol was studied using theses cultures in liquid medium under aerobic conditions, at initial concentrations of 0.25 – 2.5 mM 2-chlorophenol. Undegraded 2-chlorophenol was quantified by high-performance liquid chromatography (HPLC). Degradation rates by isolates could be determined at concentrations up to 1.5 mM. However, higher concentrations of 2-chlorophenol (2.5 mM) were inhibitory to cell growth.


INTRODUCTION
Chlorinated phenols are common environmental contaminants; they have been extensively used as biocides, mainly as wood preservatives (Abd-El-Haleem et al., 2002).Chlorinated phenols and other chlorinated phenollic compounds are also formed as by-products when chlorine is used for bleaching of pulp (Kringstad and Lindstrom, 1984) and for disinfection of drinking water and waste water containing phenols (Ahlborg and Thunberg, 1980;Detrick, 1977).
Chlorinated phenols are also formed during combustion of organic matter (Ahling and Lindskog, 1982) and as biological breakdown products of chlorophenoxyacetic *Corresponding author.E-mail: abdelhaleemm@yahoo.de.Tel: 009744852702.acid herbicides (Haggblom, 1990;Reineke and Knackmuss, 1988).A range of chlorinated organic compounds including chlorophenols can be produced by biologic chlorination as well (Neidleman and Geigert, 1986).Therefore, in the last few years, contamination of the environment by chlorinated aromatic compounds has been the subject of increased concern.
In water, chlorophenols sorb onto particulate material and, if not degraded, eventually end up in sediments.Chlorinated phenolics have been found to accumulate in freshwater and marine environments where they may attain concentrations of tens of milligrams per kilogram of dry sediment.In general, biological methods are preferable methods to treat aromatic compounds because it is economical, and there is a low possibility of the production of byproducts (Marcos et al., 1997).In this concern, several microorganisms used are usually aerobes, including Acinetobacter (Abd-El-Haleem et al., 2002), Pseudo-monas putida ( Tarık and Ermine, 2004), Ochromonas sp.(Semple and Cain, 1996), and Rhodococcus sp.(Bergauer et al., 2005).These aerobes are more efficient at degrading toxic compounds because they grow faster than anaerobes and usually transform organic compounds to inorganic compounds.
Despite the fact that microbial degradation of chlorophenols has been investigated for many years, there is still considerable interest in the metabolic capacity of bacteria able to degrade chlorophenols within indigenous microbial consortia in various ecosystems (Annachhatre and Gheewala, 1996;Solyanikova and Golovleva, 2004).Moreover, the indigenous bacteria adapted to chlorophenols in the aeration pond were proposed for application to the biological waste water treatment (Matafonova et al., 2006).Most investigations are concerned with studying pathways and enzymes involved in biodegradation of chlorophenols, using culture collection strains, or with using engineered aerobicanaerobic systems to improve biodegradability.The aim of the present work was to investigate 2-chlorophenol degrading bacteria newly isolated from different Qatari polluted soils.

Sampling enrichment culturing and soil analysis
Five soil samples were collected from different Qatari Ecosystems; one sample from Al-Ruwais, one sample from Al-Wakrah and three samples from different sites at the industrial zone at Umm-Saied city.Directly, after collection, soils were kept at 4°C until used.Later, soil samples were prepared for culturing by mixing 50 g moist weight with agitation in sterile distilled water (1:1 w/v) for 1 h at room temperature.
Analysis of the two soil samples (S1 and S2) that gave colonies after the enrichment culturing procedure was performed.The analysis was performed using the central lab facilities at Qatar University.Analysis was carried out using the inductively coupled plasma mass spectrometry ICP/Ms Series 7500.It can measure trace elements as low as one part per trillion (ppt) or quickly scan more than 70 elements to determine the composition of an unknown sample.

Biochemical analysis of the isolates
Dioxygenase activity was performed using the indigo test.Bacterial colonies were pre-grown on LB agar plates (5 g of yeast extraction, 10 g of Casein/peptone, 10 g NaCl, 15 g agar and H2O to 1 L) and then indol crystals were plated on the lid of the Petri dish.After 1 day incubation at room temperature, colonies that produce a blue color were scored positive.The activity of catalase was determined by the appearance of air bubbles after addition of a drop of 30% hydrogen peroxide solution to an overnight grown single bacterial colony.To determine the ability of isolates to hydrolyze starch, 50 µL of liquid cultures of the isolated strains were dropped on starch Petri dishes (Per liter, 3 g meat extract, 10 g starch and 14 g agar).
After one day, the inhibition zones were determined.For casine hydrolysis, 50 µL of liquid cultures of the isolated strains were dropped on casein Petri dishes (10 g casein, 14 g agar dissolved in 1 L and then autoclave).After one day, the inhibition zones were determined.In addition, the six isolated stains were subjected to classical gram staining.

Molecular analysis of the isolates
DNA extraction of all isolates was performed by using AMSHAGE DNA extraction kit (Abd-El-Haleem, 2005).To detect phenol hydroxylase DNA fragments coding for the largest subunit of multicomponent phenol hydroxylases (LmPH) were amplified by using the Applied Biosystems thermal cycler and 50 µl mixtures containing 25 µl of the 2 x PCR master mixes (Cinnagen Inc.), 100 pmol of each primer, and 50 ng of template DNA.The primers used are phe3r (5-CAGSCGRTWACCKCGCCAGAACC-3) and phe1f (5GARGGCATCAARATY-3) (Futamata et al. 2001).The PCR conditions for the two primer sets phe1f and phe3r, were as follows: step 1, 10 min of activation at 94 o C; step 2, 35 cycles consisting of 1 min at 94 o C, 1 min at 50 o C, and 1 min at 72 o C; step 3, 10 min of extension at 72 o C. The PCR products were checked by electrophoresis through 1.2% (w/v) agarose gel in TBE buffer and then staining with ethidium bromide (0.5 µg/ml: Sigma, Bornem, Belgium) and then visualized and photographed in MultiImage light cabinet (Alpha Innotech Corporation, USA).The sizes of the amplified products were assessed by comparison with a commercial weight marker.The sizes of the amplified products were assessed by comparison with a commercial weight marker (Invitrogen, Netherlands).

Comparative sequence analysis of 16S r DNA
Molecular identification of the selected isolates was performed by the amplification of 16S rDNA with eubacterial universal primers 27F and 1492R (Lane, 1991).Sequencing was performed using ABI PRISM dye terminator cycle sequencing kit with AmpliTaq DNA polymerase and an Applied Biosystems 373 DNA sequencer (Perkin-Elmer, Foster City, Calif.).The sequences were analysed using the CHECK CHIMERA and the SIMILARITY RANK programs of the Ribosomal Database Project (Altschul et al., 1990) also analysed using the BLAST program (National Centre for Biotechnology Information) to determine the closest available database sequences.Selected rDNA sequences were aligned using the Clustal W program (Shingler, 1996).Published sequences were obtained from GenBank.A phylogenetic tree was constructed using Clustal W by distance matrix analysis and the neighbour-joining method (Saitou and Nei, 1987).Phylogenetic trees were displayed using TREEVIEW (Page, 1996).

Genomic fingerprinting using REP-PCR based approach
REP-PCR (Repetitive extragenic palindromic sequence PCR (REP-PCR) Fingerprinting patterns from bacterial genomic DNA were generated with BOX1 primer (Louws, 1994).Each isolate was grown on a phenol minimal agar plate for 48 h, and a small amounts of cells from a colony were picked by a needle and sus-

Nucleotide sequence accession numbers
The nucleotide sequence data reported in this study have been deposited in the NCBI nucleotide sequence databases (GenBank) under accession numbers EF611184, EF611183, EF611182, EF611181, EF611180 and EF611179.

Growth inhibition assay
This test was performed to determine the inhibition effect of various 2-chlorophenol concentrations on the growth of the isolated bacterial strains after 24 h of incubation.Experiments were performed by inculcate 2.5 ml of overnight culture of each strain into a flask (100 ml) containing 22.5 ml of MP media supplanted individually with chlorophenol concentrations 0.25, 0.5, 1, 1.5, 2 and 2.5 mM.Cultures were incubated aerobically with shaking (150 rpm) at 35 o C. Optical densities were then measured by spectrophotometer (Jenway Ltd) at 600 nm for one week.

Determination of the biodegradation rate
Chlorophenol degradation experiments were performed in shake flasks (50 ml of MP medium containing 50 ppm 2-chlorophenol) for 96 h of incubation with free cells of each strain.To measure the degradation rates, chlorophenols were quantified each 24 h by high-performance liquid chromatography.Prior to analysis, the sam- Al-Thani et al. 2677 ples (0.3 to 0.5 ml) were acidified with 10 µL of 1 N HCl, centrifuged, and filtered (0.45 µm).Analysis was performed with a Beckman 332 LC chromatograph (Beckman Instruments, Palo Alto, Calif.) equipped with a Spherisorb C-18 column (250 by 4.6 mm; Supelco Inc., Bellefonte, Pa.), with UV detection at 280 nm, and using a solvent system of methanol (60%, vol/vol)-water (38%, vol/vol)-acetic acid (2%, vol/vol) at a flow rate of 1 ml/min.

Soil analysis
S1 and S2 soil samples which gave bacterial colonies after enrichment culturing procedure were chemically analyzed as descried above in the materials and methods section.The analysis results are presented in Table 1 at which the soil sample S2 (Al-Wakrah) recorded higher values for both heavy metals and cations content when compared to the soil sample S1 (El-Ruwais).

Biochemical analysis of the isolates
As shown in Table 2, Gram-staining revealed that all selected isolates are Gram-positive bacteria.No dioxygenase activity was recorded for all tested isolates (data not shown).Catalase and casine assays showed positive results only with isolates 6 and 3, respectively.In contrast, all tested isolates recorded positive results with the starch hydrolysis assay.Antibiotic resistant profile showed that isolates 3 and 6 are resistant to penicillin, and isolate 2 is resistant only to erythromycin.

Molecular analysis of the selected bacterial isolates
Chromosomal DNA of the bacterial isolates was extracted using AMSHAGE DNA extraction kit (Abd-El-Haleem 2005).As shown in Figure 1, the applied method was so effective and reliable that it resulted in relatively high DNA concentrations.Comparative sequence analysis of the 16S ribosomal DNA (~550 bp) of the isolates revealed that all isolates are closely related to the genus of Bacillus exhibiting similarity values ranging from 98-99% (Figure 2).Two strains QUCASBSD-1 and QUCASBSD-2 are closely related to species of Bacillus ceruse, strains QUCASBSD-4, QUCASBSD-5 and QUCASBSD-6 relate to Bacillus thuringiensis.However, only strain QUCASBSD-3 is relates to species of Bacillus licheniformis.Molecular heterogeneity among the 2chlorophenol-degrading bacteria was investigated.As shown in Figure 3, REP-PCR profiles were able to discriminate among all isolates.It was able to recognize the six isolates into two groups; the first includes isolates QUCASBSD-1, QUCASBSD-3 and QUCASBSD-6, while the second group includes strains QUCASBSD-2, QUCASBSD-4 and QUCASBSD-5, respectively.The distribution of phenol hydroxylase catabolic genes among   examined strains revealed that strains QUCASBSD-3, QUCASBSD-5 and QUCASBSD-6 yielded positive PCR amplified products of LmPH genes in the range of expect- ed sizes (~ 700 bp).No PCR amplified bands were observed with isolates QUCASBSD-1, QUCASBSD-2 and QUCASBSD-4 (Figure 4).

Biodegradation experiments
With this investigation, we demonstrated the 2chlorophenol-degradation capability of the isolated Bacillus species.The isolated Bacillus strains exhibited degradation potential for 2-chlorophenol at concentrations up to 1.5 mM in liquid medium under aerobic conditions (Figure 5).At concentrations of 2-chlorophenol between 0.25 and 1.5 mM, the Bacillus strains showed increasing rates of degradation; however, higher 2chlorophenol concentrations (2.5 mM) were inhibitory to growth.As shown in Figure 6, after four days of growth the isolates were able to degrade varying amounts of 2chlorophenol.

DISCUSSION
The present work describes the isolation of native bacterial strains from different Qatari ecosystems with the  ability to degrade one of the most important phenol derivatives (2-chlorophenol) as a sole carbon source based on culture enrichment technique.Due to its simplicity compared with the other isolation tools, it has been reported that culture enrichment methods are highly selective resulting in the isolation of a few microbial species from various natural microbial populations (Dunbar et al., 1997).
All of 2-chlorophenol-degrading bacteria described in this work were Gram-positive spore-forming rods with dimensions 1.8 -3.5 mm.They were catalase positives with exception of strain QUCASBSD-6.However, no relationships were observed between the dioxygenase test (indigo assay) and the biodegradation process.Indol conversion to indigo is considered representative of dio-Al-Thani et al. 2679 xygenase activity.The indol conversion was previously shown by aromatic ring dioxygenase from monocyclic aromatic hydrocarbon degraders (Wubbolts et al., 1994).
The phylogenetic dendrogram generated on the basis of 16S rDNA sequences revealed that bacterial isolates QUCASBSD-4, QUCASBSD-5 and QUCASBSD-6 are clearly separated into main cluster.Out of these results and the results of the soil analysis, it is possible to con-clude that each isolation sampling site has its own 2chlorophenol-degrading bacteria even if they are genetically closely related.Moreover, bacterial isolates also produced different REP-PCR fingerprint patterns.Compared with other conventional molecular biological methods, REP-PCR approach offers the advantages of ease of performance, rapid and sufficient results for species identification with small quantities of DNA (Abd-El-Haleem et al., 2002).
It is established that molecular approaches have expanded our knowledge of the diversity and distribution of microbial populations in the environment.Genes coding for catabolic enzymes such as methane monooxygenase (Henckel et al., 2000), ammonia monooxygenase (Pace et al., 1986), catechol dioxygenase (Okuta et al., 1998), and phenol hydroxylase (Watanabe et al., 1998) have also been retrieved from the environment in order to gain insight into the genetic diversity of catabolic populations.It is currently expected that such genetic information could aid in understanding and advancing bioremediation (Watanabe and Baker, 2000).
In phenol-degrading bacteria, two types of phenol hydroxylase are known, single-component and multicomponent enzymes (LmPHs) (Harayama et al., 1992).Among them, multicomponent enzymes are considered the major ones in the environment (Watanabe et al., 1998).Therefore, in the present study, we analyzed genes for the largest subunit of multicomponent phenol hydroxylases using previously designed group-specific PCR primers for LmPHs (Futamata et al., 2001).As shown in Figure 2, three bacterial isolates QUCASBSD-3, QUCASBSD-5 and QUCASBSD-6 yielded positive PCR products with phenol-degrading genes.However, these results do not mean that the other three isolates QUCASBSD-2, QUCASBSD-4 and QUCASBSD-5 are not 2-chlorophenol degraders.It is known that the degradation of phenol and its derivatives takes place aerobically through two main metabolic pathways; initiated by ortho-or meta-cleavage (Zaki, 2006).The enzymes phenol hydroxylase and catechol 1,2-dioxygenase or 2,3-dioxygenase catalyze the first and the second steps of phenol degradation, respectively (Gaal and Neujahr, 1979) which need to be covered for the isolated strains in next studies.
In conclusion, our results suggest that the bacterial community in both S1 and S2 soil samples contain strains such of genus Bacillus, which exhibit high potential for effective degradation of 2-chlorophenol under aerobic conditions.The contribution of such species, derived from contaminated sites, can be better understood by investigating their degradative potential under laboratory conditions; this serves as a major experimental strategy for understanding bioremediation processes (Demnerova et al., 2005).If the growth of these organisms can be selectively supported in the environment, they will contribute to the degradation of 2-clorophenol in a wide range of concentrations, up to 2.5 mM, and there-fore decrease the impact of pollutants.

Figure 2 .
Figure 2. Phylogenetic tree showing the relationships among isolated strains (in boldface) and published 16S rDNA sequences (their GenBank accession numbers are present in the brackets).

Table 1 .
Chemical analysis of the soil samples S1 and S2.
pended in 25 µL of MilliQ H2O.Cells were heated at 95 o C for 10 min, immediately cooled on ice, and centrifuged.The PCR was performed in 50 L (as described above).The reaction were performed in a DNA thermal cycler, 1 cycle at 95 o C for

Table 2 .
Biochemical characterization of the bacterial isolates.