Preliminary Research On Tetrodotoxin Production Of Shewanella Alga

Since widely distributed Tetrodotoxin (TTX) in marine vertebrates (e.g. fishes, amphibian), invertebrates (e.g. proboscis worm, gastropods, cephalopoda, annelid, arthropod, arrow worms, and echinoderms) and marine sediment, and various species TTX-producing bacteria have been discovered and isolated. Shewanella alga, a typical TTX-producing bacterium, has been cultured and studied on its capability of production of Tetrodotoxin. In experiment, systemically, microorganism resuscitation, purification, culture condition optimization, isolation and detection of TTX are included.Initial Shewanella alga concentration is 10-15% (w/w), 17.785μg and 7.712μg TTX were detected in bacteria cells and fermentation solution respectively at the end of culture (24~28hrs). Optimized culture conditions are: temperature 24℃~28℃ , salinity <30%o, pH 7.5-8.5, FePO4 0.01%, inoculation quantity 10-15%, aerobic and culture 24h~48h are favorable. Meantime, silica gel chromatography and TLC were first used in experiment, along with CD-180 cationic resin. Ideal results were achieved in samples preparation.In experiment, three TTX detection methods are used: ICR mouse bioassay, HPLC-PDA, and HPLC- Fluorescence detection. Bioassay method is used in determination of TTX-poisoning symptoms and estimation of TTX amount approximately. HPLC-PDA detection conditions are: reverse phase HPLC, Hypersil C18 5μm 250×4.6mm i.d. column, mobile phase: 2mmol/L sodium heptanesulfonate in 0.05mol/L phosphate buffer (NaH2PO4: Na2HPO4 is 1:1), pH: 6.8, flow rate 0.6mL/min, T. 29℃, and detected at 205nm. Method linearity is l~200mg/L and detection limit is 20ng. Another equipment detection, based on reaction of TTX can be prehydrolyzed to 2-amino-6-hydrooxyl-methyl-8-hydroxyquinazoline (C9-base)with alkali chemicals, experimental optimized conditions are: Hypersil C18 5μm 250×4.6mm i.d. column, eluted with 2mmol/L sodium heptanesulfonate in phosphate buffer, pH 6.8, reaction coiling channel 10mx0.3mm i.d., derivation temperature 100℃ and 2mol/L NaOH as derivative matter, flow rate 0.5mL/min, T. 29℃, and excitation and emission wavelength are 381nm and 505nm respectively. Method linearity is 0.5~200mg/L and detection limit is 5ng. Fluorescence is more sensitive and accurate than UV detection and accordingly recommended for TTX detection.Crystal TTX has not been isolated due to relative low production of TTX in fermentation solution, even though Shewanella alga capability and behavior of TTX production has been successfully studied. At the end of thesis, Shewanella alga and TTX knowledge and facing problems are also reviewed and discussed.