Characteristics of several bacterial isolates capable of degrading chl

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Biochemistry Lab Faculty of Bioscience and Bioengineering Prepared By Abubakr Hassan Anmar A. Mohammed Mohamed Orsod Ragheed Hussam Abdalla Hasan Abdalla Alfoqhi Mugambwa Joseph

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Characteristics of several bacterial isolates capable of degrading chloroaliphatic compound via hydrolytic decolorination

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Objective To identify bacterial isolates which can degrade haloaliphatic compound via hydrolytic decolorination

Degradation : 

Degradation BACTERIA HYDROLYTIC DECOLORINATION HALOALIPHATIC COMPOUNDS

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What is the halo aliphatic compounds ? Chemical compounds(short chain) are produced by chemical industries . Hazardous substance to animal and human( toxity and carcinogenic). Contamination resource for soil and water. uses Organic solvent . -Degreasing agent -Paints . -Pesticides . Herbicides . Intermediate for the synthesis of other organic compound

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In this study 11 bacteria strains capable of degrading 1-1,2 DCA 2- 2CPA 3- 2,3 DCPA 4- 2MCA By hydrolytic dechlorination in aerobic condition Sample are taken from:- Waste water. Rice paddy soil.

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Examination have been taken for :- Morphology. Biochemical characteristics. Degradation capabilities for Halo aliphatic hydrocarbon.

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On the basis 0f 16 S r DNA sequences , 8 different kinds of microbial specie including :- Xanthbacter flavus . Ralostonia eutorpha. Pseudomanas plecoglossicidia . Were identified

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All strains degrade MCA. UE- 2 UE-15 degrade 1,2 DCA. CA-11 degrade 2,3 DCPA .

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10 DEHALOGENATION It is the critical step in the degradation of chlorinated aliphatic . Because the reaction occurs as the first step in the degrative pathway without any regard to the mechanism of hydrolytic , oxygenatic or reductive dehalogenation

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Fig. 1. Proposed catabolic pathway of 1,2-DCA and MCA under aerobic condition. (A) 1,2-dichloroethane (1,2-DCA) (B) 2-chloroethanol (C) 2-chloroacetaldehyde (D) 2-monochloroacetic acid (2-MCA) (E) glycolic acid. DhlA, haloalkane dehalogenase; Mox, alcohol dehydrogenase; Ald, aldehyde dehydrogenase; DhlB, haloacetate dehalogenase A B C D DhIA MOX AId E DHIB H2o HCL H2o NADH2 H2o HCL

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A number of enzymes involved in degradation have been purified and characterized. Nucleotide sequences for dehalogenase have been cloned and sequenced from several bacteria . During hydrolytic dehalogenation step 1,2 DCA , 2CAA , and 2 MCA The chlorine replaced by hydroxyl group originated from water molecule under aerobic condition

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The isolation of the bacteria capable of dehalogenated aliphatic hydrocarbon Was the first carried out for Bioremediation of the contaminated chemicals

MATERIALS AND METHODS : 

Materials FeSO4.7H2O (NH4)2 SO4.7H2O NaCl Phosphate buffer (pH 7.0) Substrates Halo aliphatic hydrocarbons –source of carbon and energy and these are 1,2-dicholoroethane 2-chloropropionic 2,3-dichloropropionic acid 2-monocholoro acetic acid MATERIALS AND METHODS

Methods and materials cont. : 

Other materials Ferric ammonium sulfate Incubators Scanning electron microscope Osmium tetroxide 400ml flasks Methods Isolation and cultivation of isolates Scanning microscopy Methods and materials cont.

MATERIALS AND METHODS CONT. : 

PROCEDURES Isolation and cultivation of isolates Ten grams of samples are taken from waste waters and added to the minimal medium. a medium which contains nutrients for colony growth, generally for growing wild micro-organisms. components in the medium include:- 1µmFeSO4.7H2O 1mM (NH4)2SO4.7H2O 100µm CaCl2.2H2O 8.5µM NaCl 10mM Phosphate buffer(pH 7.0) All the above are to be contained in 400ml flasks each containing halo aliphatic hydrocarbons (source of carbon and energy) MATERIALS AND METHODS CONT.

PROCEDURES CONT. : 

PROCEDURES CONT. After pH adjustment 0.5mm of 1, 2 – dichloroethane, 2- chloropropionic acid, 2- 3 dichloroacetic acid and 1mm of 2- monochloro acetic acid are added as substrates. Under aerobic conditions, these cultures are cultivated in a shaking incubator at 30oC for 72 hours Single colonies are isolated from the medium after two times enrichment of the broth media The isolates are then examined for the hydrolytic dechlorination of chloroalihpatic compounds as in the next slide.

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After 16hrs cultivation period under aerobic conditions, the isolated strains are washed in 10mM potassium phosphate buffer twice and incubated at 30oC for 24hrs in 50mm potassium phosphate buffer that is supplemented with 0.5mM of each substrate. One ml of culture supernatant is treated with Hg (SCN)2 (0.69 g/l or 1, 0.5ml) and incubated for 15min at room temperature. The culture is then mixed with 0.5ml of 0.25M Ferric ammonium sulfate dissolved in 4.86 NHNO3.

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The concentration of releasing chloride ions is measured with a spectrophotometer ( LKB4046, Pharmacia, England) at the wavelength of 453nm By comparing the blanks with no bacterial cells, the results are evaluated. The cells from the broth agar plate are collected and prefixed with 2.5% glutaraldehyde in 100mm potassium buffer (PH 7.2 for 2hrs), and then they are post-fixed with 1% osmium tetroxide in the same buffer for 1h

Scanning electron microscopy : 

Scanning electron microscopy The cells are then dehydrated with a serial concentration (30 – 90%) ethanol every 10min and then 100% ethanol for 20min twice Substitute the cell with absolute isoamy acetate for 30min twice, and then cells are air dried. The cells are coated with gold using a sputter coater and examined with a scanning electron microscope

Amplification of 16S rDNA: : 

Amplification of 16S rDNA: PCR was used for this purpose: PCR Mixture (50 µl): 10 mM Tris-Cl (pH 8.3) 50 mM KCl 1.5 mM MgCl. 200 µM concentration of each deoxynucleotide triphosphate. Primers: 50 pM of each primer; 27F (5'-AGAGTTTGATCMTGGCTCAG-3') 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). 2.5 U of Taq DNA polymerase. A small amount of culture cells as a template source for direct PCR.

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The PCR process 95°C for 45 sec 55°C for 45 sec 72°C for 1 min 30 sec Final extension, at 72°C for 10 min

Cloning and sequencing analysis of 16S rDNA: : 

Cloning and sequencing analysis of 16S rDNA: Ligation of the amplified 1.5 kb PCR products with PGEM-T vector. Transformation E. coli XL1-blue Plasmid rescue and DNA purification purified plasmid Labelled Primer Sequencing Methods DNA sequences Ribosomal Database Project Sequence Match and Similarity Matrix programs The 16S rDNA sequences of the isolates

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PGEM-T vector:

Biochemical characteristics : 

Biochemical characteristics API 20 NE was used for this purpose. - The API 20 NE strip consists of 20 microtubes containing dehydrated substrates. - Catalase activity of the bacterial isolates was tested with 3% H2O2. - Oxidase activity was tested with 1% tetramethyl-p-phenylendiamine 2HCl. - The resistance to antibiotics was examined using disc-diffusion methods on the agar medium.

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PAI 20NE Process: Inoculation of a saline bacterial suspension which reconstitutes the media Incubation at the proper temperature The bacteria grow if they are capable of utilizing the corresponding substrate The color changes

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- The reactions are read according to the Reading Table and the identification is obtained by referring to the Analytical Profile Index or using the identification software. Ex. The API 20NE result for Pseudomonas aeruginosa Disc-diffusion method: incubation

Identification and homology on the basis of 16S rDNAsequence : 

Identification and homology on the basis of 16S rDNAsequence belong to Ancyclobacter sp. and Xanthobacter flavus. In resent studies, Pseudomonas, Comamonas, and Burkholderia species have been isolated as soil microorganisms which can degrade a variety of chloroaliphatic compounds as well as many aromatic compounds

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Phylogeny is a different and newer method of classification that detects differences in microorganisms based upon genetic characteristics. Its evolutionary relationship among gene and organism . Showing us which gene and organism are related .