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Production of a novel cold-induced extracellular biopolymer by Pseudomonas fluorescens BM07, its structural characterization and environmental applications

슈도모나스 플루오레슨스 BM07에 의한 저온에서의 새로운 바이오폴리머의 과분비생산,

캄비즈 (Kambiz Akbari Noghabi, 대학원 응용생명과학부)

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This thesis describes the production and characterization of a novel cold-induced biopolymer produced by Pseudomonas fluorescens BM07. The current study was also undertaken to assess the effect of physico-chemical factors on the production of BM07 biopolymer. We were particularly interested in relat...
This thesis describes the production and characterization of a novel cold-induced biopolymer produced by Pseudomonas fluorescens BM07. The current study was also undertaken to assess the effect of physico-chemical factors on the production of BM07 biopolymer. We were particularly interested in relating the occurrence of polymer production with ambient temperature and its structural characterization and environmental applications. Pseudomonas fluorescens BM07 was induced to excrete a sticky extracellular biopolymer by decreasing the temperature down to as low as 10ºC. Maximum production of the cold induced biopolymer was obtained when cells were grown aerobically at 10ºC and its synthesis was inhibited at 30ºC. A reverse correlation between temperature and biopolymer production was shown. BM07 biopolymer was compositionally unique and compositional analysis of protein fraction as the major constituent (95 % w/w) revealed that it was composed mainly of hydrophobic (42 mol %), and less frequently of charged (32 mol %) and polar (28 mol %) amino acids. Analysis by High performance anion-exchange chromatography with pulse amperometric detector (HPAEC-PAD) showed that glycosyl moiety comprised a small fraction of biopolymer (~ 5 % w/w), composed of glucosamine and galactosamine in a molar ratio of 9:1. Culture conditions generally did not affect the amino acid and monosaccharide compositions. However, the use of gluconic acid and glycerol as carbon source resulted in the formation of a biopolymer which contained glucose in addition to glucosamine and galactosamine. Fourier-transform infrared spectroscopy (FT-IR) demonstrated the presence of carboxyl, amine, hydroxyl and methoxyl functional groups in exopolymer. The dried biopolymer had low solubility in water and required at least 1 week agitation to be solubilized but when it left to stand without agitation colloidal suspension is formed. BM07 biopolymer showed high ion capacity with particular preference to uptake mercury and cadmium (70% and ~ 45%) respectively. Electron microscopy analysis revealed that the exopolymeric substances form a matrix in which bacterial particles are clumped together within the flocs. Exopolymer looks like a sheath surrounding each bacterial cell and extending out (fingerlike projection) toward the adjacent cells, helping them to make bacterial aggregation. Biopolymer showed adhesive properties in which cells can not be seen at all and were presumed to be covered by a layer of biopolymer matrix. The flocculating efficiency of the biopolymer was considerable and correspondingly increased with growth phase and cultivation time at 10 ºC especially in logarithmic phase (from 96 h to 144 h) in which biopolymer mainly released into the culture medium. Addition of various cations such as: Al^3+, Fe^3+, Mn^2+ and Ca^2 as co-flocculants to purified biopolymer had significant synergistic effect on flocculating rate. In the light of results obtained, extracellular biopolymer was involved in the aggregation of bacteria into flocs. All the factors examined including carbon and nitrogen sources, temperature, detergent, bioelement, pH and salinity seemed to influence biopolymer production efficiency.
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Abstract = I
Content = III
List of Tables = V
...
Abstract = I
Content = III
List of Tables = V
List of Figures = VI
I. Introduction = 1
1. History = 1
2. Extracellular biopolymer chemistry = 5
3. Extracellular biopolymer function = 6
3.1. Adhesion to surface = 6
3.2. Aggregation process = 9
3.3. Removal of metal ions = 10
3.4. Waste water treatment = 12
4. Extracellular biopolymer production = 15
5. Other functions = 16
6. EPS in marine environment = 18
6.1. EPS in particulate form = 18
6.2. EPS as transparent exopolymer particle = 20
7. Objective & Scope = 20
II. Materials & Methods = 21
1. Microorganism & Culture condition = 21
2. Biopolymer extraction = 22
3. Effect of detergents on polymer production = 23
4. Extracellular biopolymer analysis = 23
5. Analysis of the monosaccharide composition = 24
6. Flocculating activity assay = 25
7. Heavy metal absorption = 25
8. Physical analysis of biopolymer = 26
9. X-ray diffraction = 27
10. Scanning electron microscopy (SEM) = 27
11. Gel permeation chromatography (GPC) = 27
12. SDS-PAGE analysis = 28
III. Results = 30
1. Effect of carbon and nitrogen sources = 30
2. Effect of couple-substrate feeding = 33
3. Effect of detergents on biopolymer production = 37
4. Effect of bioelements & salinity on polymer production = 39
5. Effect of temperature and initial pH = 42
6. Isolation of biopolymer and its solution properties = 45
7. Analysis of glycosyl moiety of BM07biopolymer = 50
8. Preliminary structural analysis of dried biopolymer = 52
9. X-ray diffraction = 58
10. Scanning electron microscopy (SEM = 59
11. Determination of flocculating activity = 65
12. Heavy metal biosorption = 68
IV. Discussion = 70
V. References = 74
Acknowledgments = 88