Measurement of proteolysis in natural waters

Microbiological proteolysis in Lake Champlain water was measured in situ and in vitro by the spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder. Water samples sterilized by microfiltration were never proteolytic. In situ proteolysis was found to be very dependent upon water temperature (1 to 23 degrees C). No measurable activity was observed below 4 degrees C. The in vitro proteolysis rate at 20 degrees C was found to be 2.3 times the rate at 15 degrees C and 6 times the rate at 10 degrees C. Water taken from beneath the ice-covered lake throughout the winter and tested in the laboratory at 20 degrees C was found to show an increasing proteolytic potential during the winter months. The highest activity was obtained as the ice broke up in early spring. Microbiological proteolysis in water from Burlington Harbor was often four times that found in center lake water. In most experiments proteolysis was inhibited completely by 2 mug of Cu and inhibited 67% by 0.75 mug/ml. Proteolysis was markedly stimulated by 20 to 40 mug of Casitone or Casamino Acids per ml. The predominant bacteria growing in the proteolysis flasks were species of Pseudomonas and Flavobacterium. Pure cultures of Pseudomonas required traces of Casitone, Casamino Acids, or yeast extract for proteolysis of hide powder azure, whereas those of Flavobacterium did not. The requirement could not be met by a mixture of 21 amino acids and eight vitamins.