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Isolation and Indentification of Oproturon Degrading Strains:Degradation Characteristic and Application

Author: SunJiQuan
Tutor: LiShunPeng
School: Nanjing Agricultural College
Course: Microbiology
Keywords: phenylurea herbicides isoproturon Sphingomonas sp. Y57 Sphingobium sp. YBL1 Sphingobium sp. YBL2 Sphingobium sp. YBL3 biodegradationand bioremediation Azospirillum xianwuii sp. nov
CLC: X172
Type: PhD thesis
Year: 2008
Downloads: 14
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Abstract


Five strains of bacteria (designated as Y57, YBL1, YBL2, YBL3, CA6respectively) capable of degrading isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, or capable of degrading aniline which was thought as the critical metabolite in the degradation of isoproturon in environment, were isolated from the soils of three herbicide plants. Strain Y57was isolated from the soil collected from Changzhou, Strain YBL1was isolated from the soil collected from Nanjing and other two strains were isolated from the soil collected from Suzhou. Based on the comparative analysis of16S rRNA gene, phenotypic and biochemical characterization, strain Y57was identified as Sphingomonas sp., other three strains were identified as Sphingobium sp.. The optimum conditions for strains YBL1, YBL2and YBL3growth were pH6.0, and temperature30℃, while the optimum condition for strain Y57growth was pH7.0and temperature35℃. All these strains can not tolerate2.5%NaCl. Growth rate was related positively to oxygen availability.Strain CA6was isolated from soil which was collected from Suzhou. On the basis of polyphasic taxonomy, morphological characterization, Biolog analysis, Cellular fatty acid and16S rRNA gene sequence analysis, the isolate represent a new species within the genus Azospirillum. Its closest phylogenetic neighbors, as deduced by16S rRNA gene analysis, are Azospirillum rugosum AFH-6T, Azospirillum brasilense DSM1690T and Azospirillum Hpoferum DSM1691r with96.6,96.3and96.2%sequence similarity respectively. The chemotaxonomic characteristics of CA6(G+C content is70.5mol%and18:1ω7c as the major cellular fatty acid, and14:03-OH and16:03-OH as the major hydroxy fatty acids) were consistent with the genus Azospirillum. Cells of CA6are rods and curve,0.7-0.9μm in width and1.5-2.4μm in length, Gram-negative and motile with single polar flagellum. Optimum growth occurs at37℃and at pH values between6.0and7.0. They have a respiratory type of metabolism, grow well on Dextrin, a-D-Glucose, Maltose, Mono- Methyl-Succinate, β-Hydroxy Butyric Acid, L-Alanyl-glycine. For this species, the name Azospirillum xianwuii sp. nov. is suggested, with strain CA6(=CCTCC AB208097=JCM15509) as the type strain.In the degradation test, more than99%of the initial isoproturon (50mg·l-1, Y57was30mg·l-), added to the medium as the sole carbon source, were degraded by these strains at the inoculation rate of107cells per mili liter within20hours. Strains showed optimum degradation activity at pH7.0, which dropped sharply above pH9.0or below pH6.0, and the strain lost the ability to degrade isoproturon at pH5.0; the optimum temperature for isoproturon degradation by these strains was30℃, however these strains can degrade isoproturon between20℃to35℃as well. Supplementing the medium with glucose considerably enhanced isoproturon degradation rate; but slow down degradation rate by strain Y57in MSM medium, the addition of Cu2+, Ni2+at the rate of1mmol·l-1inhibited the capability of these strains to degrade isoproturon, while the Mg2+in the same quantity enhanced the isoproturon degradation rate under the same conditions. These four strains also showed the ability to remove the residues of other phenylurea herbicides such as chlorotoluron, diuron and fluometuron in mineral salt culture medium. Strain YBL1was found capable of degrading both, dimethylurea-substituted herbicides and methoxy methyl phenylurea herbicide i.e. linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea). Strain Y57was capable of degrading propail in addition to isoproturon and chlorotoluron. The concentration of isoproturon (within the chemical solubility in water) had no apparent effect on the degradation rate.Strains YBL1, YBL2and YBL3degraded isoproturon significantly (>80%) in Magan soil, and Flvo-Aquic soil within two days, while in Red soil the degradation activity was very poor. The degradation rate of other phenylurea herbicides, such as chlorotoluron and diuron was also observed higher in Magan soil. These three strains also have ability to degrade isoproturon in fresh soil (non-sterilized), but the degradation rate was slower in fresh soil than in sterilized soil, and the degradation rate of isoproturon by these strain was related positively to the soil moisture (10%-40%, v:v). The most adaptable temperature for these strains degrading isoproturon in soil was35℃, which was higher than the optimum temperature for isoproturon degradation in MSM The degradation rate was related positively to inoculation quantity, when the inoculum was below10CFU per gram soil, these strains were unable to degrade the isoproturon after two days inoculation. Glucose suplementation can enhance the degradation rate of isoproturon in soil, while the addition of NH4NO3showed the reverse results.MDBPU (3-(4-isopropylphenyl)-1-methylurea) was detected in the catabolic products of isoproturon, and the chemical3-(3-chloro-4-methylphenyl)-1-methylurea, was detected in the degradation of chlorotoluron by YBL2, analysed by HPLC-MS. The catechol, which was thought as the critical metabolite in the process of phenyl-ring cleavage of most aromatic hydrocarbon, was detected in the beginning of aniline degradation by these strains. Moreover the cis, cis-muconic acid, the first metabolite after phenyl-ring cleavage, was also detected in the degradation products of aniline by HPLC.Additon of strain Y57can reduce the effect of chlorotoluron residue on the growth of maize, and addition of strains YBL1, YBL2and YBL3respectively can clean up the isoproturon residue in laboratory experiments, and protected the rape from prototoxitity of isoproturon. Using culture of YBL3in a field, which was contaminated by chlorotoluron as a result of repeated use located at Taixing, can clean up the residue of chlorotoluron in soil. All these results indicate that these strains can be used for bioremediation of phenylurea herbicides contamination.

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