DECOMPOSITION OF SURFACTANTS BY BACTERIA 69 Broth(100ml) I Centrifuged I • Supernatant PPt(CelI) -Freeze-drying Freeze-dried Matters 200mg Dissolved in M/15 Phosphate Buffer Crude Enzyme A (Extracellular Enzyme) --Washed with sterile water -Dehydrated with Acetone (pHS.2) •Dehydrated with Ethyl Ether PPt.(Dried Cell) Cell(100mg) Suspended in M/15 Phosphate Buffer (10ml) --Ultrasonic Disintegration --Centrifuged ! i Supernatant PPt. Crude Enzyme B (Endocellular Enzyme) Figure 1. Preparation of crude enzyme THE PREPARATION METHOD OF CRUDE ENZYME The crude enzyme was prepared according to Figure 1. The broth culture was centrifuged and the supernatant liquid was freeze-dried. 200 mg of the freeze-dried matter was dissolved in M/15 phosphate buffer solution. This enzyme was extracellu- lar. On the other hand, the cells were washed and dehydrated to obtain dried cells. These dried cells were disintegrated by ultrasonic wave and centrifuged. The supernatant liquid was crude enzyme B. This enzyme was endocellular. THE MEASUREMENT OF ENZYME ACTIVITY Five grams of polysorbate-20 was homogeneously dissolved in 10 ml of M/t5 phosphate buffer solution (pH 8.2) and l0 ml distilled water. Five milliliters of the above solution was transferred into a lO0-ml Erlenmeyer flask with a glass stopper. Two ml crude enzyme was added to it and allowed to react with polysorbate-20 at 37øC for 2 hr with shaking. After 2 hr, 30 ml of 99.5% ethyl alcohol was added to stop the reaction and titrated with 0.05N NaOH solution by using phenolphthalein as an indicator. The activity of esterase was expressed as the volume (milliliters) of 0.05N NaOH consumed. Blank values were obtained by adding ethyl alcohol immediately after the crude enzyme was added and titrating. THE MEASUREMENT OF INACTIVATION OF ENZYME BY A U.V. LAMP A solution of 20 ml crude enzyme was placed into a quartz vessel (2 cm by 2 cm by 4 cm) and covered with a thin quartz plate to prevent evaporation of water. The enzyme

70 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table•1 Viable Counts in Storage Tanks Viable Count/ml CPS Number Predominant Strain Tank Capacity Nu•• of CPS Nutrient Species Group Strain Laboratory A 10oe 8.6X 10 4 7.4X 103 12 6 W-6 77801 B 15koe 1.5X •'05 8X 10 1900 4 W-6 78231 C 30koe 4.3 X •04 1.2 X 10 4 3.6 2 W-6 78718 Plant D 5 koe 4.3 X 103 2.0 X 10 2 22 5 W-6 78755 E 8k•, 1.3X102 2X10 6.5 4 W-13 781014 F 5koe 1X10 10 1 W-6 781215 *Now improved to 102/ml was irradiated with a U.V. lamp from a distance of 2 cm. There 2 ml of crude enzyme solution was taken out of the vessel and its activity was measured. RESULTS AND DISCUSSION BACTERIA The viable counts and bacterial flora of deionized water Bacteria in the range of 10 to 100,000 per ml were found in the storage tanks of deionized water (Table II). Four to five species were isolated from one tank, the group 10 6- E 10 5- 103 •) 1'2 1'8 Culture Time Sterile Deionized water //"'••X.. -'"'""'"•x Distilled water ß 'tilled Water 2'4 Hrs. Figure 2. Growth curve of indigenous bacteria in purified water