Pseudomonas aeruginosa Mac 436 was found to produce simultaneously three phenazine pigments identified as pyocyanine, phenazine-1-carboxylic acid, and oxychlororaphine. Production of these pigments on various media indicated a wide variation in yields depending on the composition of the media, but satisfactory yields of all three pigments were obtained. A scheme was developed for separation and assay of the pigments from the culture liquor. Details of production, isolation, assay, and identification are given.

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A B S T R A C T A neutral extracellular glucan ([a]nZ3 +18g0) was produced i n 127, yield b y Pullularia pullulans (de Bary)The yeast-like fungus Pullularia pullulans produces a mixture of glucose-containing extracellular polrsaccharides (I). I t has been shown (2, 3) that one of the polysaccharides formed by the fungus from glucose as the carbon source is a linear glucan ("pullulan") having [a], +192" and coinposed of approximately 300 a-D-glucopyranose units linked -(1 + 4) and (1 -+ 6) in the ratio 3:2. Bouveng et al. (4) have examined the polysaccharides produced by Pullularia pullulans from various sugar substrates, and have found that a sucrose medium yielded a linear glucan having [a], +190° and consisting of more than 250 units linked a ( l -+ 4) and a ( 1 -+ 6) in the ratio 2:l. A small amount (1-27,) of glucose remaining after periodate oxidation of the polysaccharide suggested that (1 -+ 3) linkages might be present, although this was not supported by methylation data. No evidence for the presence of (1 -+ 3) linkages ilT the polysaccharide produced from glucose was reported in earlier work (2, 3). The present publication reports the results obtained from partial acid hydrolysis and froin periodate oxidation of the glucan produced by Pullularia pullulans grown on glucose.The growth conditions of the organism and the method of recovery used for the preparation of the polj~saccharide in the present work favored isolation of the water-soluble glucan to the exclusion of the more insoluble jelly-like polysaccharide which adheres to the mycelium (1). The polysaccharide showed a single symmetrical peak on electrophoresis in borate and acetate buffers, gave no precipitate with Cetavlon, and showed no carboxyl absorption in its infrared spectrum. This evidence showed that the polysaccharide was homogeneous and contained no acid groups. Bouveng et al. (4) found that yields of polysaccharide were increased and that formation of uronic acid and other hexose units was diminished when the organism was grown on glucose rather than other sugar substrate (except for sucrose).Infrared spectroscopy of the polysaccharide showed strong absorption a t 850 cm-l,

published studies on chromogenic bacteria, related to Escherichia coli, which they renamed Escherichia auresceus (Parr) comb. nov. In listing reasons for suggesting this classification they referred to metabolic products of these cultures, as reported in a personal communication concerning a study carried out in this laboratory. The data to which they referred are given in this report on the effects of pH on the products of anaerobic metabolism of glucose by pigmented and nonpigmented strains of Escherichia coli. MATERIALS AND METHODS Three cultures of Escherichia aurescenq labeled Ba, No.

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Aspergillus niger PRL 558 was used in the production of starch saccharifying enzymes. The culture was grown on 100 ml. of medium in 500 ml. Erlenmeyer flasks agitated and aerated on a rotary shaker at 35° C. Quantitative analyses for amylase, maltase, and limit dextrinase were determined on the culture filtrates. Variations in the specific type of carbohydrate source affected the yield of amylase markedly, and of maltase to a lesser extent. The production of limit dextrinase is the least dependent on the carbohydrate source. Maltose or compounds constituted of maltose units are essential for producing a maximum yield of amylase. The yields of the enzymes are related to the degree of the availability of the nitrogen source. Highest enzyme yields are obtained with hydrolyzed protein. However, the production of amylase and maltase is further stimulated by adding inorganic nitrogen compounds such as ammonium nitrate and sodium nitrate as a supplementary nitrogen source. The amounts of maltase and amylase obtained are controlled also by varying the fermentation time as well as the carbohudrate and protein content of the medium.

Cell-free extracts of Aerobacter aerogenes grown on a medium containing yeast extract, glucose, and salts, oxidized D-α-fructoheptose (2-C-hydroxymethyl-D-glucose), glucose, gluconate, and other hexoses and pentoses. The enzymes were in a particulate fraction and were difficult to purify. This cell-free enzyme system required magnesium or certain other divalent metal ions as activators and was not stimulated by any coenzyme tested. Phosphorylation did not appear to be involved. The pH-stability and pH-activity curves for the crude enzyme preparation were plotted and the Kmvalue for D-α-fructoheptose determined. The same enzyme system is apparently involved for all substrates and appears to be specific for sugars having the same configuration as glucose at the second and fourth carbon atoms.

Use of coumarin by soil fungi has been studied. The route of coumarin degradation was suggested to proceed first by reduction to dihydrocoumarin and then by hydration to melilotic acid.