Studies on the Bioemulsification of Diesel by the Rhizobial Exopolysaccharides

• Main Authors: Wen-Chung Lin, 林文中
• Other Authors: Chieh-Chen Huang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/49320608557370323738

碩士 === 國立中興大學 === 生命科學系所 === 94 === Sorption and sequestration of xenobiotic compounds within the soil matrix are critical processes affecting contaminant mobility, toxicity, and persistence. Slow desorption and release from the soil matrix to the aqueous phase represents a long-term contaminant source and hinders remediation efforts. Both synthetic and biological surfactants have been shown to enhance the apparent aqueous solubility of non-polar organic compounds resulting in increased bioavailability and biodegradation. However, there are also reports, which suggest that some synthetic surfactants inhibit biodegradation. This inhibition is generally attributed to toxicity or reductions in bioavailability due to partitioning of contaminant into surfactant micelles. Some microorganisms secrete biosurfactants under certain incubation conditions. Members of genus Rhizobia (an important genus of symbiotic bacteria) are being used extensively as inoculants for legumes. In addition to the improvements in plant N nutrition, they have characteristic exo-secretions. To study the surfactant activities of the Rhizobial exo-secretions and their emulsification ability 17 different strains of Rhizobia were tested in the present study under different growth conditions. Effect of pH (pH4 ~ pH10), temperature (20 ~ 40℃), agitation (90 rpm ~ 180 rpm), carbon source (glucose, sucrose, mannose, lactose, galactose, fructose, arabinose, maltose and xylose), nitrogen source ((NH4)2SO4, NH4Cl, NaNO3, KNO3, Glycine, L-Glutamic acid, L-Asparagine and Casamino acid), and vitamins (D-biotin, Ascorbic acid and Ca-pantothenate) were tested to optimize the ideal conditions for the increased production of exopolyasscharide by Rhizobia. The biosurfactnats produced were tested for their ability to emulsify diesel oil. Among the 16 Rhizobial species tested the biosurfactant produced by Rhizobium sp. CC-13H showed a comparatively higher diesel emulsification index of 57.1% than the others. The secreted exopolysaacharide was water-soluble but insoluble in alcohol. After freeze-drying the exopolyasscharide yield was 6.82 g L-1 (0.68%) from a 66 hr. grown medium. The major finding of this study was that the tested Rhizobial strain produced an exopolysaacharide, which showed an emulsification index of 53.3% . Quantification of the compound was performed by Phenol-sulfuric acid method wherein; hydrolysis of polysaccharides in presence of 95% sulfuric acid yields monosaccharides, and upon dehydration in presence of phenol an aldehyde-carbohydrate compound was formed. This compound showed absorption maximum at 490 nm. The amount of exopolyasscharide production was 398 μg mL-1 for the tested Rhizobium sp. CC-13H strain under optimum incubation temperature, pH and shaking rate with 30℃, 6.8 and 150 rpm respectively. Regulation of shaking rate between 90 rpm and 180 rpm, at pH 6 showed adaptation to produce the bioemuslifier. This strain produced 411 μg mL-1 exopolyasscharide with 59.2% emulsification index at 150 rpm ,while 460 μg mL-1 exopolyasscharide with 68% emulsification index was observed at 125 rpm. Different carbon and nitrogen source showed direct influence on the exopolyasscharide production. Among the tested carbon and nitrogen sources, the strain CC-13H produced a maximum of 591 μg mL-1 exopolyasscharide with emulsification index of 81% when the YEM media was supplemented with 1.5% mannitol and 592 μg mL-1 exopolyasscharide with 80% emulsification index for diesel oil when YEM was supplemented 1.5% mannitol and 0.1% NaNO3. Vitamins also showed to regulate the exopolyasscharide production. The strain CC-13H produced 658 μg mL-1 exopolysaacharide with 94% emulsification index when YEM media was supplemented with 1.5% mannitol, 0.1% NaNO3 and 0.001 g L-1 riboflavin. This component showed an emulsification index of 97.8% when tested against kerosene. Compared to Surfactin of Bacillus subtilis, with 80%, Rhamnolipid of Pseudomonas aeruginosa with 75% and Emulsan of Acinetabacter calcoaceticus with 74% emulsion index for kerosene, the present compound showed a higher emulsion index (97.8%) . From the results Rhizobia sp. CC-13H strain can be employed for the industrial scale biosurfactant production along with its use in bioremediation of oil polluted sites.