Development of Methods for Immobilization and Usages of Immobilized Recombinant Bioluminescent Strain, Pseudomonas Putida Mt-2 KG1206, Stored by Deep-Freezing

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 (KNO 3 ) 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.Keywords: Bioluminescence; Biomonitoring; Deep-freezing; Immobilization; Recombinant Significance and Impact of the StudyThis study indicated that type of immobilization matrix, cell conditions (subcultured or centrifuged pellets), matrix (beads) numbers, volume of reconstitution solution, and thawing time are important factors on bioluminescence activity of reconstituted culture, preserved by deep freezing. Overall, this investigation demonstrated the ability of immobilized bacteria on glass beads, preserved by deep freezing, for the monitoring of a specific group of environmental pollutants using a stimulant KNO 3 , suggesting the potential method for preliminary application in a field-ready portable bioassay.