Abstract
Deep-freezing process can be used practically for environmental monitoring. However, for the organism to be useful in environmental applications, the strain of bacteria exhibits a high intensity of activity following reconstitution after deep-freezing. An overall protocol for immobilization and reconstitution of recombinant bioluminescent bacteria, preserved by deep-freezing was investigated for future biomonitoring. Tested strain KG1206 has ability to produce bioluminescence in the presence of toluene analogs and those intermediates. Immobilization using glass beads with subcultured cells showed better results than other conditions, such as alginate beads or centrifuged cell pellets. Beads number, thawing time, and amount of reconstitution solution influence on bioluminescence activity. Among tested conditions, followings were appeared as optimum conditions: working volume 15 mL of serum vials, approximately 20-30 glass beads per vial, 3 h thawing time, and 2-5 mL reconstitution solution. Potassium nitrate (KNO3) greatly stimulates the low bioluminescence activity of immobilized strain, preserved by deep freezing, in the range of 2.5 to 21 times of untreated one.
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