(511x) Isolation and Identification of a Strain of Klebsiella Oxytoca with 2,4-Dichlorophenol-Degrading Activity

Isolation and Identification of a Strain of Klebsiella oxytoca with 2,4-Dichlorophenol-Degrading Activity   Wang Leo Qiang ^1*, Wang Jianing ², Zhu Zhichen³, Ma Peisheng ⁴

1.*
College of
Material Science and Chemical Engineering, Tianjin University of Science and Technology, TEDA, Tianjin, 300457,
China, wang_q@tust.edu.cn   

2. Biology Research Institute, Shandong
Academy of
Science, Jinan, 250014,
China       

3. Department of Material Science,
Tianjin College of Urban Construction, Tianjin, 300072,
China

4.
College of
Chemical Engineering,
Tianjin University, Tianjin, 300072,
China

Phenolic compounds are widely distributed in the environment from various industrial as well

 as natural sources, for example chlorinated phenolic compounds are specifically utilized as insecticides, herbicides, detergents, solvents, wood preservatives and antimicrobial agents. Biological methods are preferable methods to treat aromatic compounds because it is economical, and there is a low possibility of the production of byproducts^[1]. Several microorganisms used are usually aerobes, including Pseudomonas putida ^[2-3], Ochrobactrum sp.^[4], , Rhodococcus sp. ^[5], Burkholderia sp.^[6].  These aerobes are more efficient at degrading toxic compounds because they grow faster than anaerobes and usually transform organic compounds to inorganic compounds. Except for these reports above, there is few published information available in the literature regarding the existence of aromatic ring cleavage activities within different species of genus Klebsiella.

In this paper, a new dichlorophenol-degrading strain was isolated from return activated sludge used in the biological treatment of waste-water of Jizhuangzi wastewater plant in
Tianjin. The sludge enriched on phenol and 2,4-dicholorophenol was used as a source of chlorophenol-degrading bacteria. The enrichment was maintained for 24 months with bimonthly transfers of 20% inocula to fresh revised aerobic mineral medium . Cells were grown in a stirred chemostat at room temperature under stationary conditions, Approximately 10 mM phenol 0.2mM 2,4-dichlorophenol as the carbon source was added every week, pH 6.5-7.0^[7].

We confirmed the identification by sequencing the entire 16s rRNA gene. The 16s rDNA was amplified using a pair of forward (5¡¯- AGAGTTTGATCCTGGCTCAG-3¡¯) and reverse (5¡¯-TACGGCTACCTTGTTACGACT -3¡¯) primers following genomic DNA extraction  and the corresponding PCR . 16s rDNA containing plasmid vectors were constructed using the pGEM-T Easy vector and used to transform E. coli cells for the nucleotide sequence analysis of the selected clones. The 16s rDNA sequences presents the strain that can be identified as Klebsiella oxytoca.  This is first time to report about the Klebsiella oxytoca could be applied to degradating 2,4-dichlorophenol.

To clarify the level of dichlorophenol biodegradation in strain Klebsiella oxytoca, this activity was investigated.  Kinetic parameters for 2,4-dichlorophenol degradation by cells were determined by measuring 2,4-dichlorophenol disappearance rates. 2,4-Dichlorophenol were determined by monitoring changes in 2,4-dichlorophenol concentration, using a modified extended Kalman filter assay^[8-9].The most important factor of influence reaction rate is temperature, for example, 50 mg/L 2,4-dichlorophenol can be reduced to 1.0 mg/L by Klebsiella oxytoca that 36 hours is needed at 32¡æ, but 72 h is needed at 25¡æ.

In conclusion, a new 2,4-dichlorophenol-degrading strain, Klebsiella oxytoca , was isolated from phenol-activated sludge in
Tianjin. Phenotypic characteristics and phylogenetic analysis indicated the strain belong Klebsiella oxytoca.

Reference

1.       Alasdair H Neilson£¬An Environmental Perspective on the Biodegradation of Organochlorine Xenobiotics[J]£¬International Bioakterioration & Biodegradation,1996,3:21-24

2.       Tarık Abuhamed, Emine Bayraktar, Kinetics model for growth of Pseudomonas putida F1 during benzene, toluene and phenol biodegradation[J], Process Biochem,2004,39:983"C988

3.       Gurusamy Annadurai, Juang Ruey-Shin, Microbiological degradation of phenol using mixed liquors of Pseudomonas putida and activated sludge[J], Waste Manage£¬2002, 22: 703"C710

4.       Velasco, J., Romero, C., Lopez-Goni, I., Leiva, J., Diaz, R., and Moriyon, I.: Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp. [J], Int. J. Syst. Bacterial,1998, 48: 759-768

5.       Margesin R, Fonteyne PA, Low-temperature biodegradation of high amounts of phenol by Rhodococcus sp. and basidiomycetous yeasts[J], Res Microbiol£¬2005, 156: 68"C75

6.        Wang Q, Ma P-S, Kinetics of phenol degradation by streptococcus, Proceedings of 9^th CEST ,
Greece, 2005,1622-1626

7.        Wang Q, Ma P-S, Nonlinear Kalman filter simultaneous determination of phenol and o-chlorophenol[J],  Spectroscopy and spectrum analysis, 2006, 26: 899-904

8.        Wang Q, Ma P-S, Derivative Kalman filter simultaneous determination of phenol ,o-chlorophenol and 2,4dichlorophenol[J],  Spectroscopy and spectrum analysis, 2007, 27: 560-564